Salt form of a human histone methyltransferase EZH2 inhibitor

ABSTRACT

Provided herein is N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl (tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide hydrobromide. Also provided herein is a particular polymorph form of this compound.

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 15/837,390, filed Dec. 11, 2017, now U.S. Pat. No.10,245,269, which is a continuation application of U.S. patentapplication Ser. No. 15/199,522, filed Jun. 30, 2016, now U.S. Pat. No.9,872,862, which is a continuation application of U.S. patentapplication Ser. No. 14/394,431, filed Oct. 14, 2014, now U.S. Pat. No.9,394,283, which is a U.S. National Phase Application of InternationalApplication No. PCT/US2013/036193, filed Apr. 11, 2013, which claimspriority to, and the benefit of, U.S. Provisional Application No.61/624,215, filed Apr. 13, 2012, the entire contents of each of whichare incorporated herein by reference in their entireties.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The contents of the text file named “EPIZ-010N01US SeqenceListing.txt”,which was created on May 3, 2016 and is 1 KB in size, are herebyincorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

More than 1.6 million people are estimated to be diagnosed with cancerin 2012. For example, the most common type of cancer in women is breastcancer, and this disease is responsible for one of the highest fatalityrates of all cancers affecting females. The current treatment of breastcancer is limited to total, or partial, mastectomy, radiation therapy,or chemotherapy. Almost 230,000 of cancer cases in 2012 will be breastcancer, which will result in an estimated 40,000 deaths. See, Siegel etal., Ca Cancer J Clin 2012; 62:10-29.

A number of cancer deaths are caused by blood cancers includingleukaemias, myelomas, and lymphomas. In 2012, almost 80,000 of cancercases will be lymphomas, which will result in an estimated 20,000deaths.

Radiation therapy, chemotherapy, and surgery are the primary methods ofcancer treatment. However, these therapies are most successful only whenthe cancer is detected at an early stage. Once cancer reachesinvasive/metastatic stages, lines of invading cells or metastasizingcells can escape detection, thus resulting in relapses, which requiresthe use of therapy that is highly toxic. At this point, both the cancercells and the patient's unaffected cells are exposed to the toxictherapy, resulting, among other complications, a weakening of the immunesystem.

As such, there remains a need in the art for new methods for treatingcancer, such as breast cancer or lymphomas, in a patient.

SUMMARY OF THE INVENTION

Accordingly, provided herein isN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidehydrobromide:

Also provided herein is a particular polymorph form ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidehydrobromide (“Polymorph A,” or “Polymorph A ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidehydrobromide”). As described herein, the hydrobromide salt providedherein, as well as Polymorph A, exhibit physical properties that can beexploited in order to obtain new pharmacological properties, and thatmay be utilized in drug substance and drug product development.

In one embodiment, the hydrobromide is crystalline. In anotherembodiment, the hydrobromide is substantially free of impurities. Inanother embodiment, the hydrobromide is a crystalline solidsubstantially free of amorphousN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidehydrobromide.

In another aspect, provided herein is a pharmaceutical compositioncomprising the hydrobromide described above, and a pharmaceuticallyacceptable carrier or diluent.

In one aspect, the hydrobromide described above is prepared using amethod comprising combiningN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidewith hydrobromic acid.

Polymorph A ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidecan be defined according to its X-ray powder diffraction pattern.Accordingly, in one embodiment, the polymorph exhibits an X-ray powderdiffraction pattern having one or more characteristic peaks expressed indegrees 2-theta at about 3.9+/−0.3 degrees, about 17.5+/−0.3 degrees,and about 22.0+/−0.3 degrees 2-theta. In another embodiment, thepolymorph exhibits an X-ray powder diffraction pattern havingcharacteristic peaks expressed in degrees 2-theta at about 3.9+/−0.3degrees, about 17.5+/−0.3 degrees, and about 22.0+/−0.3 degrees 2-theta.In still another embodiment, the polymorph exhibits an X-ray powderdiffraction pattern having characteristic peaks expressed in degrees2-theta at about 3.9+/−0.3 degrees, 10.1+/−0.3 degrees, 14.3+/−0.3degrees, 17.5+/−0.3 degrees, 18.7+/−0.3 degrees, 20.6+/−0.3 degrees,20.9+/−0.3 degrees, 21.8+/−0.3 degrees, 22.0+/−0.3 degrees, 23.3+/−0.3degrees and 23.6+/−0.3 degrees 2-theta. In still another embodiment, thepolymorph exhibits an X-ray powder diffraction pattern substantially inaccordance with FIG. 1. In another embodiment, the polymorph exhibits anX-ray powder diffraction pattern substantially in accordance with Table1.

Polymorph A can also be defined according to its differential scanningcalorimetry thermogram. In one embodiment, the polymorph exhibits adifferential scanning calorimetry thermogram having a characteristicpeak expressed in units of ° C. at a temperature of 255+/−5° C. In anembodiment, the polymorph exhibits a differential scanning calorimetrythermogram substantially in accordance with FIG. 3.

In one aspect, Polymorph A is prepared using a method comprisingcombiningN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidewith hydrobromic acid.

In another aspect, provided herein is a method of recrystallizingPolymorph A, which comprises the following steps: (a) dissolvingPolymorph A in a first solvent, and (b) adding a second solvent, suchthat said polymorph is recrystallized. In one embodiment, the firstsolvent is ethanol, and the second solvent is MTBE. In anotherembodiment, the method comprises (a) dissolving Polymorph A in ethanol,(b) heating the mixture, (c) adding MTBE to the mixture, forming aprecipitate comprising said polymorph, and filtering the precipitatesuch that said polymorph is recrystallized.

In still another aspect, provided herein is a pharmaceutical compositioncomprising Polymorph A, and a pharmaceutically acceptable carrier ordiluent.

Also provided herein is a method of treating cancer comprisingadministering to a subject in need thereof a therapeutically effectiveamount of the hydrobromide compound described above, Polymorph A, or apharmaceutical composition comprising either of these compounds. Avariety of cancers can be treated, including non-Hodgkin's lymphoma orbreast cancer.

In another aspect, provided herein is a method of inhibiting the histonemethyltransferase activity of EZH2 in a subject in need thereofcomprising administering to the subject an effective amount of thehydrobromide compound described above, Polymorph A, or a pharmaceuticalcomposition comprising either of these compounds.

In still another aspect, provided herein is a method of inhibiting thehistone methyltransferase activity of EZH2 in vitro comprisingadministering the hydrobromide compound described above, or Polymorph A.

Also provided herein is the use of the hydrobromide compound describedabove, Polymorph A, or a pharmaceutical composition comprising either ofthese compounds, for the preparation of a medicament for the treatmentof cancer in a subject in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the X-ray powder diffraction pattern of Polymorph A(monohydrobromide).

FIG. 2 depicts the X-ray powder diffraction pattern the dihydrobromideof Compound I.

FIG. 3 depicts the differential scanning calorimetry thermogram ofPolymorph A.

FIG. 4 depicts the dynamic vapor sorption of Polymorph A, whichdemonstrates the low hygroscopicity of this compound.

FIG. 5 depicts HPLC analysis of Polymorph A over three days at anelevated temperature. Polymorph A produced minimal impurities over thistime.

FIG. 6 depicts the dynamic vapor sorption of the sodium salt of CompoundI, which demonstrates the significant hygroscopicity of this compound.

FIG. 7 depicts the dynamic vapor sorption of the hemisulfate salt ofCompound I, which demonstrates that this compound has moderately highhygroscopicity.

FIG. 8 shows differential scanning calorimetry data of themonohydrochloride salt of Compound I, which indicates that this compoundis poorly crystalline.

FIG. 9 depicts the X-ray powder diffraction pattern of syntheticintermediate 5.

FIG. 10 depicts the X-ray powder diffraction pattern of Polymorph B.

FIG. 11 depicts the X-ray crystal structure of the monohydrobromide ofCompound I.

FIG. 12A and FIG. 12B show the results from in vivo studies of thehydrobromide of Compound I in a SUDHL10 mouse xenograft; FIG. 12A is aplot of the growth of the SUDHL10 xenograft tumors over time for thedifferent treatment groups and FIG. 12B illustrates mean tumor weight onday 28.

FIG. 13 shows the global HSK27me3 methylation in SUDHL tumors from micetreated with the hydrobromide of Compound I for 28 days.

FIG. 14A shows the tumor re-growth over time for different treatmentgroups after dosing stopped on day 28.

FIG. 14B illustrates survival percentages for different treatmentgroups.

FIG. 15 shows the anti-cancer effect of the hydrobromide of Compound Ion a lymphoma mouse xenograft model.

FIG. 16 shows the anti-cancer effect of the hydrobromide of Compound Ion a lymphoma mouse xenograft model.

FIG. 17 depicts the X-ray powder diffraction pattern of syntheticintermediate 2.

FIG. 18A and FIG. 18B depict (A) the X-ray powder diffraction pattern ofthe trihydrochloride salt of Compound I and (B) the dynamic vaporsorption of the monohydrochloride salt of Compound I, which demonstratesthe significant hygroscopicity of this compound.

DETAILED DESCRIPTION OF THE INVENTION HBr Salt Form and Polymorph Form A

Provided herein isN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidehydrobromide:

As used herein, “Compound I” refers toN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide.The hydrobromide of Compound I can be used to inhibit the histonemethyltransferase activity of EZH2, either in a subject or in vitro. Thehydrobromide of Compound I can also be used to treat cancer in a subjectin need thereof.

Compound I can be protonated at one or more of its basic sites, such asthe morpholine, disubstituted aniline, and/or pyridone moieties.Accordingly, in certain embodiments, provided herein is themonohydrobromide, dihydrobromide, or trihydrobromide of Compound I. Inone embodiment, provided herein is the monohydrobromide of Compound I.When the compound is the monohydrobromide, the compound may beprotonated at any basic site. In a non-limiting embodiment, Compound Iis protonated at the nitrogen of the morpholino substituent, providing amonohydrobromide of Compound I having the following structure:

This particular monohydrobromide can be referred to as“4-((3′-(((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)carbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methyl-[1,1′-biphenyl]-4-yl)methyl)morpholin-4-iumbromide.” FIG. 11 depicts the X-ray crystal structure of this particularsalt form.

The hydrobromide of Compound I has a number of advantageous physicalproperties over its free base form, as well as other salts of the freebase. In particular, the hydrobromide of Compound I has lowhygroscopicity compared to other salt forms of Compound I. For acompound to be effective in therapy, it is generally required that thecompound be minimally hygroscopic. Drug forms that are highlyhygroscopic may be unstable, as the drug form's dissolution rate maychange as it is stored in settings with varying humidity. Also,hygroscopicity can impact large-scale handling and manufacturing of acompound, as it can be difficult to determine the true weight of ahygroscopic active agent when preparing a pharmaceutical compositioncomprising that agent. The hydrobromide of Compound I has a lowhygoscopicity compared to other salt forms of Compound I. As such, itcan be stored over appreciable periods, and not suffer from detrimentalchanges in, for example, solubility, density, or even chemicalcomposition.

In addition to the above advantages, the hydrobromide of Compound I canbe produced in a highly crystalline form, which is useful in thepreparation of pharmaceutical formulations, and will improve generalhandling, manipulation, and storage of the drug compound. In a preferredembodiment, the crystalline form of the hydrobromide of Compound I is ina form referred to as “Polymorph A.”

The ability of a substance to exist in more than one crystal form isdefined as polymorphism; the different crystal forms of a particularsubstance are referred to as “polymorphs.” In general, polymorphism isaffected by the ability of a molecule of a substance to change itsconformation or to form different intermolecular or intra-molecularinteractions, particularly hydrogen bonds, which is reflected indifferent atom arrangements in the crystal lattices of differentpolymorphs. In contrast, the overall external form of a substance isknown as “morphology,” which refers to the external shape of the crystaland the planes present, without reference to the internal structure.Crystals can display different morphology based on different conditions,such as, for example, growth rate, stirring, and the presence ofimpurities.

The different polymorphs of a substance may possess different energiesof the crystal lattice and, thus, in solid state they can show differentphysical properties such as form, density, melting point, color,stability, solubility, dissolution rate, etc., which can, in turn,affect the stability, dissolution rate and/or bioavailability of a givenpolymorph and its suitability for use as a pharmaceutical and inpharmaceutical compositions.

Polymorph A is highly crystalline, and displays low hygroscopicity.Also, this polymorph can be obtained reproducibly, and slight changes incrystallization conditions do not result in different crystal forms.

Access to different polymorphs of the hydrobromide of Compound I isdesirable for a number of reasons. One such reason is that individualpolymorphs can incorporate different impurities, or chemical residues,upon crystallization. For example, impurities can be removed during theprocess of converting Compound I into Polymorph A.

Without wishing to be bound by theory, polymorph forms exhibitingcompact crystal shapes possess advantages in terms of ease of filtrationand ease of flow. Polymorph A exhibits a compact crystal shape thattherefore possesses these advantages.

In certain embodiments, Polymorph A is identifiable on the basis ofcharacteristic peaks in an X-ray powder diffraction analysis. X-raypowder diffraction, also referred to as XRPD, is a scientific techniqueusing X-ray, neutron, or electron diffraction on powder,microcrystalline, or other solid materials for structuralcharacterization of the materials. In one embodiment, Polymorph Aexhibits an X-ray powder diffraction pattern having one or morecharacteristic peaks expressed in degrees 2-theta at about 3.9+/−0.3degrees, about 17.5+/−0.3 degrees, and about 22.0+/−0.3 degrees 2-theta.In another embodiment, the polymorph exhibits an X-ray powderdiffraction pattern having characteristic peaks expressed in degrees2-theta at about 3.9+/−0.3 degrees, about 17.5+/−0.3 degrees, and about22.0+/−0.3 degrees 2-theta.

In one embodiment, Polymorph A exhibits an X-ray powder diffractionpattern having at least 5 characteristic peaks expressed in degrees2-theta at about 3.9+/−0.3 degrees, 10.1+/−0.3 degrees, 14.3+/−0.3degrees, 17.5+/−0.3 degrees, 18.7+/−0.3 degrees, 20.6+/−0.3 degrees,20.9+/−0.3 degrees, 21.8+/−0.3 degrees, 22.0+/−0.3 degrees, 23.3+/−0.3degrees and 23.6+/−0.3 degrees 2-theta. In another embodiment, PolymorphA exhibits an X-ray powder diffraction pattern having at least 6characteristic peaks expressed in degrees 2-theta at about 3.9+/−0.3degrees, 10.1+/−0.3 degrees, 14.3+/−0.3 degrees, 17.5+/−0.3 degrees,18.7+/−0.3 degrees, 20.6+/−0.3 degrees, 20.9+/−0.3 degrees, 21.8+/−0.3degrees, 22.0+/−0.3 degrees, 23.3+/−0.3 degrees and 23.6+/−0.3 degrees2-theta. In yet another embodiment, Polymorph A exhibits an X-ray powderdiffraction pattern having at least 7 characteristic peaks expressed indegrees 2-theta at about 3.9+/−0.3 degrees, 10.1+/−0.3 degrees,14.3+/−0.3 degrees, 17.5+/−0.3 degrees, 18.7+/−0.3 degrees, 20.6+/−0.3degrees, 20.9+/−0.3 degrees, 21.8+/−0.3 degrees, 22.0+/−0.3 degrees,23.3+/−0.3 degrees and 23.6+/−0.3 degrees 2-theta. In anotherembodiment, Polymorph A exhibits an X-ray powder diffraction patternhaving at least 8 characteristic peaks expressed in degrees 2-theta atabout 3.9+/−0.3 degrees, 10.1+/−0.3 degrees, 14.3+/−0.3 degrees,17.5+/−0.3 degrees, 18.7+/−0.3 degrees, 20.6+/−0.3 degrees, 20.9+/−0.3degrees, 21.8+/−0.3 degrees, 22.0+/−0.3 degrees, 23.3+/−0.3 degrees and23.6+/−0.3 degrees 2-theta. In still another embodiment, Polymorph Aexhibits an X-ray powder diffraction pattern having at least 9characteristic peaks expressed in degrees 2-theta at about 3.9+/−0.3degrees, 10.1+/−0.3 degrees, 14.3+/−0.3 degrees, 17.5+/−0.3 degrees,18.7+/−0.3 degrees, 20.6+/−0.3 degrees, 20.9+/−0.3 degrees, 21.8+/−0.3degrees, 22.0+/−0.3 degrees, 23.3+/−0.3 degrees and 23.6+/−0.3 degrees2-theta. In yet another embodiment, the polymorph exhibits an X-raypowder diffraction pattern having at least 10 characteristic peaksexpressed in degrees 2-theta at about 3.9+/−0.3 degrees, 10.1+/−0.3degrees, 14.3+/−0.3 degrees, 17.5+/−0.3 degrees, 18.7+/−0.3 degrees,20.6+/−0.3 degrees, 20.9+/−0.3 degrees, 21.8+/−0.3 degrees, 22.0+/−0.3degrees, 23.3+/−0.3 degrees and 23.6+/−0.3 degrees 2-theta.

In still another embodiment, the polymorph exhibits an X-ray powderdiffraction pattern having characteristic peaks expressed in degrees2-theta at about 3.9+/−0.3 degrees, about 14.3+/−0.3 degrees, about18.7+/−0.3 degrees, about 23.3+/−0.3 degrees, and about 23.6+/−0.3degrees 2-theta.

In still another embodiment, the polymorph exhibits an X-ray powderdiffraction pattern having characteristic peaks expressed in degrees2-theta at about 3.9+/−0.3 degrees, about 10.1+/−0.3 degrees, about14.3+/−0.3 degrees, about 17.5+/−0.3 degrees, about 18.7+/−0.3 degrees,about 20.6+/−0.3 degrees, about 20.9+/−0.3 degrees, about 21.8+/−0.3degrees, about 22.0+/−0.3 degrees, about 23.3+/−0.3 degrees and about23.6+/−0.3 degrees 2-theta. In yet another embodiment, the polymorphexhibits an X-ray powder diffraction pattern substantially in accordancewith FIG. 1. In another embodiment, the polymorph exhibits an X-raypowder diffraction pattern substantially in accordance with the 2-thetavalues listed in Table 1.

As used herein, the term “about”, when used in reference to a degree2-theta value refers to the stated value +/−0.3 degrees 2-theta.

Pharmaceutical compositions comprising Polymorph A can be identified bycomparison of the compositions' X-ray powder diffraction patterns to anX-ray powder diffraction pattern of Polymorph A. It will be appreciatedthat pharmaceutical compositions comprising Polymorph A may exhibitnon-identical X-ray powder diffraction patterns as compared to an X-raypowder diffraction pattern of pure Polymorph A.

In certain embodiments, Polymorph A is identifiable on the basis of acharacteristic peak observed in a differential scanning calorimetrythermogram. Differential scanning calorimetry, or DSC, is athermoanalytical technique in which the difference in the amount of heatrequired to increase the temperature of a sample and reference ismeasured as a function of temperature. In one embodiment, Polymorph Aexhibits a differential scanning calorimetry thermogram having acharacteristic peak expressed in units of ° C. at a temperature of about255+/−5° C. In another embodiment, Polymorph A exhibits a differentialscanning calorimetry thermogram having a single endothermic peakobserved at the temperature range of 250-255° C. In another embodiment,Polymorph A exhibits a differential scanning calorimetry thermogramsubstantially in accordance with FIG. 3.

In certain embodiments, Polymorph A may contain impurities. Non-limitingexamples of impurities include undesired polymorph forms, or residualorganic and inorganic molecules such as solvents, water or salts. In oneembodiment, Polymorph A is substantially free from impurities. Inanother embodiment, Polymorph A contains less than 10% by weight totalimpurities. In another embodiment, Polymorph A contains less than 5% byweight total impurities. In another embodiment, Polymorph A containsless than 1% by weight total impurities. In yet another embodiment,Polymorph A contains less than 0.1% by weight total impurities.

In certain embodiments, Polymorph A is a crystalline solid substantiallyfree of amorphous Compound I hydrobromide. As used herein, the term“substantially free of amorphous Compound I hydrobromide” means that thecompound contains no significant amount of amorphous Compound Ihydrobromide. In certain embodiments, at least about 95% by weight ofcrystalline Polymorph A is present. In still other embodiments of theinvention, at least about 99% by weight of crystalline Polymorph A ispresent.

In another embodiment, Polymorph A is substantially free from PolymorphB.

The salt of the invention, and its crystal form Polymorph A, can befound together with other substances or can be isolated. In someembodiments, the salt of the invention, or its crystal form, issubstantially isolated. By “substantially isolated” is meant that thesalt or its crystal form is at least partially or substantiallyseparated from the environment in which it was formed or detected.Partial separation can include, for example, a composition enriched inthe salt of the invention. Substantial separation can includecompositions containing at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 97%, or at least about 99% by weight of the hydrobromideof Compound I and Polymorph A. Methods for isolating compounds and theirsalts are routine in the art.

Both the hydrobromide of Compound I and Polymorph A can occur as anyreasonable tautomer, or a mixture of reasonable tautomers. As usedherein, “tautomer” refers to one of two or more structural isomers thatexist in equilibrium and are readily converted from one isomeric form toanother. Examples include keto-enol tautomers, such asacetone/propen-2-ol, and the like. The hydrobromide of Compound I andPolymorph A can have one or more tautomers and therefore include variousisomers, i.e., pyridin-2(1H)-one and the corresponding pyridin-2-ol. Allsuch isomeric forms of these compounds are expressly included in thepresent invention.

Preparation of HBr Salt Form and Polymorph A

The hydrobromide of Compound I, as well as Polymorph A, can be preparedusing known techniques. Conventionally, a salt form is prepared bycombining in solution the free base compound and an acid containing theanion of the salt form desired, and then isolating the solid saltproduct from the reaction solution (e.g., by crystallization,precipitation, evaporation, etc.). Other salt-forming techniques can beemployed.

Scheme 1 below outlines a particular embodiment for the production ofthe free base of Compound I, as well as the hydrobromide of Compound I.Briefly, methyl 3-amino-5-bromo-2-methylbenzoate (1) is reacted withdihydro-2H-pyran-4(3H)-one under reductive amination conditions to formmethyl 5-bromo-2-methyl-3-((tetrahydro-2H-pyran-4-yl)amino)benzoate (2)in step 1. In step 2, reductive amination is again used to form5-bromo-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methylbenzoate (3).This compound is then reacted under Suzuki coupling conditions in step 3to form methyl5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxylate(4), which is hydrolyzed to the corresponding acid (5) in step 4. Instep 5, acid (5) is reacted under amide coupling reaction conditionswith 3-(aminomethyl)-4,6-dimethyl-dihydro-pyridin-2(1H)-onehydrochloride to form Compound I.

As shown, Compound I can then be reacted with aqueous HBr to form thehydrobromide of Compound I.

The synthesis described above has a number of advantages. For example,it utilizes a number of intermediates that can be prepared incrystalline forms that can be isolated. By using crystallineintermediates, minimal purification techniques (e.g., chromatography)are necessary, leading to an overall improved yield of final Compound I.

Accordingly, provided herein is intermediate compound 1 in crystallineform. In another embodiment, provided herein is intermediate compound 2in crystalline form. FIG. 17 shows an X-ray powder diffraction patternof crystalline compound 2. In still another embodiment, the intermediatecompound 5 is crystalline. FIG. 9 shows an X-ray powder diffractionpattern of crystalline compound 5. In other embodiments, compounds 2and/or 5 are produced in substantially pure form without the use ofchromatography. It will be appreciated by the skilled artisan that thecrystallization of intermediates does not necessarily proceedeffortlessly or efficiently.

Also provided herein is a method of preparingN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidecomprising reacting5-(Ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)[1,1′-biphenyl]-3-carboxylicacid (5) with a salt of3-(aminomethyl)-4,6-dimethyl-dihydro-pyridin-2(1H)-one. In oneembodiment of this method, (5) is in crystalline form.

Compound I can be reacted with aqueous HBr in the presence of anappropriate solvent to form Polymorph A, a particular crystal form ofthe hydrobromide. In an embodiment, Compound I is reacted with aqueousHBr in the presence of ethanol and ethyl acetate to form Polymorph A.

Once the polymorph is prepared, it can be recrystallized, using the samesolvent (or solvents) that were used to prepare the polymorph, or adifferent solvent (or solvents), to produce a composition that hasincreased crystallinity. In general, Polymorph A can be recrystallizedby dissolving the polymorph in one or more solvents, optionally heating,followed by an optional cooling step, and then isolating the crystalstructure, through, e.g., a filtering step. After the polymorph isinitially dissolved in the first solvent (or combination of solvents),an additional, different solvent can be added at any point in theprocess (before or after heating, before or after cooling, etc.) toproduce the desired crystal structure. For example, a first solvent canbe used to dissolve the polymorph compound, and then a second solvent(e.g., an anti-solvent) can be added to cause the polymorph toprecipitate from solution. In an embodiment, water is added to the firstsolvent to aid in dissolving the polymorph.

Non-limiting examples of solvents that can be used for therecrystallization of Polymorph A are as follows: methanol, ethanol,ethyl acetate, methyl tert-butyl ether, water, isopropyl alcohol,tetrahydrofuran, acetone, acetonitrile, and 2-methyltetrahydrofuran, aswell as combinations thereof. Non-limiting examples of solventcombinations that are useful for the recrystallization of Polymorph Aare (solvent and anti-solvent, wherein water can be added to the firstsolvent to aid in dissolving the polymorph): methanol/water and ethylacetate, isopropyl alcohol/water and ethyl acetate,tetrahydrofuran/water and ethyl acetate, acetone and ethyl acetate,acetonitrile/water and ethyl acetate, ethanol/water and methyltert-butyl ether, isopropyl alcohol/water and methyl tert-butyl ether,ethanol/water and tetrahydrofuran, isopropyl alcohol/water and acetone,and ethanol/water and ethyl acetate. In particular embodiments, thesolvent combinations are methanol/water and ethyl acetate, isopropylalcohol/water and ethyl acetate, ethanol/water and2-methyltetrahydrofuran, and methanol/2-methyltetrahydrofuran. In oneaspect, Polymorph A is prepared using a method comprising combiningN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidewith hydrobromic acid.

In another aspect, provided herein is a method of recrystallizingPolymorph A, which comprises the following steps: (a) dissolvingPolymorph A in a first solvent, and (b) adding a second solvent, suchthat said polymorph is recrystallized. In one embodiment, the firstsolvent is ethanol, and the second solvent is MTBE. In anotherembodiment, the method comprises (a) dissolving Polymorph A in ethanol,(b) heating the mixture, (c) adding MTBE to the mixture, forming aprecipitate comprising said polymorph, and filtering the precipitatesuch that said polymorph is recrystallized.

Pharmaceutical Compositions

In another aspect, provided herein is a pharmaceutical compositioncomprising the hydrobromide of Compound I, and a pharmaceuticallyacceptable carrier or diluent. Also provided herein is a pharmaceuticalcomposition comprising Polymorph A, and a pharmaceutically acceptablecarrier or diluent.

The term “pharmaceutical composition” includes preparations suitable foradministration to mammals, e.g., humans. When the compounds of thepresent invention are administered as pharmaceuticals to mammals, e.g.,humans, they can be given per se or as a pharmaceutical compositioncontaining, for example, 0.1% to 99.9% (more preferably, 0.5 to 90%) ofactive ingredient in combination with a pharmaceutically acceptablecarrier.

The compounds described herein (i.e., the hydrobromide of Compound I andPolymorph A) can be combined with a pharmaceutically acceptable carrieraccording to conventional pharmaceutical compounding techniques. As usedherein, “pharmaceutically acceptable carrier” may include any and allsolvents, diluents, or other liquid vehicle, dispersion or suspensionaids, surface active agents, isotonic agents, thickening or emulsifyingagents, preservatives, solid binders, lubricants and the like, as suitedto the particular dosage form desired. Remington's PharmaceuticalSciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton,Pa., 1980) discloses various carriers used in formulating pharmaceuticalcompositions and known techniques for the preparation thereof. Exceptinsofar as any conventional carrier medium is incompatible with thecompounds such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutical composition, its use is contemplatedto be within the scope of this invention. Some examples of materialswhich can serve as pharmaceutically acceptable carriers include, but arenot limited to, sugars such as lactose, glucose and sucrose; starchessuch as corn starch and potato starch; cellulose and its derivativessuch as sodium carboxymethyl cellulose, ethyl cellulose and celluloseacetate; powdered tragacanth; malt; gelatine; talc; excipients such ascocoa butter and suppository waxes; oils such as peanut oil, cottonseedoil; safflower oil, sesame oil; olive oil; corn oil and soybean oil;glycols; such as propylene glycol; esters such as ethyl oleate and ethyllaurate; agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen free water; isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

Furthermore, the carrier may take a wide variety of forms depending onthe form of the preparation desired for administration, e.g. oral,nasal, rectal, vaginal, parenteral (including intravenous injections orinfusions). In preparing compositions for oral dosage form any of theusual pharmaceutical media may be employed. Usual pharmaceutical mediainclude, for example, water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents, and the like in the case of oral liquidpreparations (such as for example, suspensions, solutions, emulsions andelixirs); aerosols; or carriers such as starches, sugars,microcrystalline cellulose, diluents, granulating agents, lubricants,binders, disintegrating agents and the like, in the case of oral solidpreparations (such as for example, powders, capsules, and tablets).

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, tocopherols, and the like; and metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Pharmaceutical compositions comprising the compounds may be formulatedto have any concentration desired. In some embodiments, the compositionis formulated such that it comprises at least a therapeuticallyeffective amount. In some embodiments, the composition is formulatedsuch that it comprises an amount that would not cause one or moreunwanted side effects.

Because the crystalline form of the hydrobromide of Compound I is moreeasily maintained during its preparation, solid dosage forms are apreferred form for the pharmaceutical composition of the invention.Solid dosage forms for oral administration, such as capsules, tablets,pills, powders, and granules, are particularly preferred. If desired,tablets may be coated by techniques known to those in the art.

Pharmaceutical compositions include those suitable for oral, sublingual,nasal rectal, vaginal, topical, buccal and parenteral (includingsubcutaneous, intramuscular, and intravenous) administration, althoughthe most suitable route will depend on the nature and severity of thecondition being treated. The compositions may be conveniently presentedin unit dosage form, and prepared by any of the methods well known inthe art of pharmacy. In certain embodiments, the pharmaceuticalcomposition is formulated for oral administration in the form of a pill,capsule, lozenge or tablet. In other embodiments, the pharmaceuticalcomposition is in the form of a suspension.

The compounds provided herein are suitable as an active agent inpharmaceutical compositions that are efficacious particularly fortreating EZH2-associated disorders, especially cancer. Thepharmaceutical composition in various embodiments has a pharmaceuticallyeffective amount of the hydrobromide of Compound I or Polymorph A, alongwith other pharmaceutically acceptable excipients, carriers, fillers,diluents and the like.

A therapeutically or pharmaceutically “effective amount” is an amount ofa compound (the hydrobromide of Compound I or Polymorph A), that whenadministered to a patient, ameliorates a symptom of a disease orcondition, e.g., prevent the various morphological and somatic symptomsof cancer. In an example, an effective amount of the hydrobromide ofCompound I or Polymorph A is the amount sufficient to treat cancer in asubject. The amount can vary depending on such factors as the size andweight of the subject, the type of illness, or the particular compoundof the invention. The amount of the hydrobromide of Compound I orPolymorph A that constitutes an “effective amount” will vary dependingon the compound, the disease state and its severity, the age of thepatient to be treated, and the like. The effective amount can bedetermined routinely by one of ordinary skill in the art having regardto their knowledge and to this disclosure.

The regimen of administration can affect what constitutes apharmaceutically effective amount. The hydrobromide of Compound I orPolymorph A, and compositions comprising either of these compounds, canbe administered to the subject either prior to or after the onset of adisease. Further, several divided dosages, as well as staggered dosagescan be administered daily or sequentially, or the dose can becontinuously infused, or can be a bolus injection. Further, the dosagescan be proportionally increased or decreased as indicated by theexigencies of the therapeutic or prophylactic situation.

Methods of Treatment

Compounds of the present invention (i.e., the hydrobromide of CompoundI, as well as Polymorph A) inhibit the histone methyltransferaseactivity of EZH2 or a mutant thereof and, accordingly, in one aspect ofthe invention, certain compounds disclosed herein are candidates fortreating, or preventing certain conditions and diseases. The presentinvention provides methods for treating conditions and diseases thecourse of which can be influenced by modulating the methylation statusof histones or other proteins, wherein said methylation status ismediated at least in part by the activity of EZH2. Modulation of themethylation status of histones can in turn influence the level ofexpression of target genes activated by methylation, and/or target genessuppressed by methylation. The method includes administering to asubject in need of such treatment, a therapeutically effective amount ofa compound of the present invention.

The disorder in which EZH2-mediated protein methylation plays a part canbe cancer or a precancerous condition. The present invention furtherprovides the use of a compound of the present invention (i.e., thehydrobromide of Compound I, as well as Polymorph A) in the treatment ofcancer or precancer the course of which can be influenced by modulatingEZH2-mediated protein methylation, or, for the preparation of amedicament useful for the treatment of such cancer or pre-cancer.Exemplary cancers that may be treated include lymphomas, includingnon-Hodgkin lymphoma, follicular lymphoma (FL) and diffuse large B-celllymphoma (DLBCL); melanoma; and leukemia, including CIVIL. Exemplaryprecancerous condition includes myelodysplastic syndrome (MDS; formerlyknown as preleukemia).

In still another embodiment, provided herein is a method of treating alymphoma comprising administering to the subject in need thereof aneffective amount of the hydrobromide of Compound I.

In yet another embodiment, provided herein is a method of treating alymphoma comprising administering to a subject in need thereof aneffective amount of Polymorph A.

The present invention also provides methods of protecting against adisorder in which EZH2-mediated protein methylation plays a part in asubject in need thereof by administering a therapeutically effectiveamount of compound of the present invention (i.e., the hydrobromide ofCompound I, as well as Polymorph A) to a subject in need of suchtreatment. The disorder can be cancer, e.g., cancer in whichEZH2-mediated protein methylation plays a role. The present inventionalso provides the use of compound of the present invention (i.e., thehydrobromide of Compound I, as well as Polymorph A) for the preparationof a medicament useful for the prevention of a cell proliferativedisorder associated, at least in part, with EZH2-mediated proteinmethylation.

The compounds of this invention can be used to modulate protein (e.g.,histone) methylation, e.g., to modulate histone methyltransferase orhistone demethylase enzyme activity. At least some of the compounds ofthe invention can be used in vivo or in vitro for modulating proteinmethylation. Histone methylation has been reported to be involved inaberrant expression of certain genes in cancers, and in silencing ofneuronal genes in non-neuronal cells. At least some compounds describedherein are suitable candidates for treating these diseases, i.e., todecreases methylation or restores methylation to roughly its level incounterpart normal cells.

Compounds that are methylation modulators may be used for modulatingcell proliferation. For example, in some cases excessive proliferationmay be reduced with agents that decrease methylation, whereasinsufficient proliferation may be stimulated with agents that increasemethylation. Accordingly, diseases that may be treated by the compoundsof the invention can include hyperproliferative diseases, such as benigncell growth and malignant cell growth.

As used herein, a “subject in need thereof” is a subject having adisorder in which EZH2-mediated protein methylation plays a part, or asubject having an increased risk of developing such disorder relative tothe population at large. A subject in need thereof can have aprecancerous condition. Preferably, a subject in need thereof hascancer. A “subject” includes a mammal. The mammal can be e.g., a humanor appropriate non-human mammal, such as primate, mouse, rat, dog, cat,cow, horse, goat, camel, sheep or a pig. The subject can also be a birdor fowl. In one embodiment, the mammal is a human.

As used herein, the term “cell proliferative disorder” refers toconditions in which unregulated or abnormal growth, or both, of cellscan lead to the development of an unwanted condition or disease, whichmay or may not be cancerous. Exemplary cell proliferative disorders thatmay be treated with the compounds of the invention encompass a varietyof conditions wherein cell division is deregulated. Exemplary cellproliferative disorder include, but are not limited to, neoplasms,benign tumors, malignant tumors, pre-cancerous conditions, in situtumors, encapsulated tumors, metastatic tumors, liquid tumors, solidtumors, immunological tumors, hematological tumors, cancers, carcinomas,leukemias, lymphomas, sarcomas, and rapidly dividing cells. The term“rapidly dividing cell” as used herein is defined as any cell thatdivides at a rate that exceeds or is greater than what is expected orobserved among neighboring or juxtaposed cells within the same tissue. Acell proliferative disorder includes a precancer or a precancerouscondition. A cell proliferative disorder includes cancer. In one aspect,the methods provided herein are used to treat or alleviate a symptom ofcancer or to identify suitable candidates for such purposes. The term“cancer” includes solid tumors, as well as, hematologic tumors and/ormalignancies. A “precancer cell” or “precancerous cell” is a cellmanifesting a cell proliferative disorder that is a precancer or aprecancerous condition. A “cancer cell” or “cancerous cell” is a cellmanifesting a cell proliferative disorder that is a cancer. Anyreproducible means of measurement may be used to identify cancer cellsor precancerous cells. Cancer cells or precancerous cells can beidentified by histological typing or grading of a tissue sample (e.g., abiopsy sample). Cancer cells or precancerous cells can be identifiedthrough the use of appropriate molecular markers.

Exemplary non-cancerous conditions or disorders that may be treatedusing one or more compounds of the present invention include, but arenot limited to, rheumatoid arthritis; inflammation; autoimmune disease;lymphoproliferative conditions; acromegaly; rheumatoid spondylitis;osteoarthritis; gout, other arthritic conditions; sepsis; septic shock;endotoxic shock; gram-negative sepsis; toxic shock syndrome; asthma;adult respiratory distress syndrome; chronic obstructive pulmonarydisease; chronic pulmonary inflammation; inflammatory bowel disease;Crohn's disease; psoriasis; eczema; ulcerative colitis; pancreaticfibrosis; hepatic fibrosis; acute and chronic renal disease; irritablebowel syndrome; pyresis; restenosis; cerebral malaria; stroke andischemic injury; neural trauma; Alzheimer's disease; Huntington'sdisease; Parkinson's disease; acute and chronic pain; allergic rhinitis;allergic conjunctivitis; chronic heart failure; acute coronary syndrome;cachexia; malaria; leprosy; leishmaniasis; Lyme disease; Reiter'ssyndrome; acute synovitis; muscle degeneration, bursitis; tendonitis;tenosynovitis; herniated, ruptures, or prolapsed intervertebral disksyndrome; osteopetrosis; thrombosis; restenosis; silicosis; pulmonarysarcosis; bone resorption diseases, such as osteoporosis;graft-versus-host reaction; Multiple Sclerosis; lupus; fibromyalgia;AIDS and other viral diseases such as Herpes Zoster, Herpes Simplex I orII, influenza virus and cytomegalovirus; and diabetes mellitus.

Exemplary cancers that can be treated using one or more compounds of thepresent invention include, but are not limited to, adrenocorticalcarcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer,anorectal cancer, cancer of the anal canal, appendix cancer, childhoodcerebellar astrocytoma, childhood cerebral astrocytoma, basal cellcarcinoma, skin cancer (non-melanoma), biliary cancer, extrahepatic bileduct cancer, intrahepatic bile duct cancer, bladder cancer, urinarybladder cancer, bone and joint cancer, osteosarcoma and malignantfibrous histiocytoma, brain cancer, brain tumor, brain stem glioma,cerebellar astrocytoma, cerebral astrocytoma/malignant glioma,ependymoma, medulloblastoma, supratentorial primitive neuroectodermaltumors, visual pathway and hypothalamic glioma, breast cancer, bronchialadenomas/carcinoids, carcinoid tumor, gastrointestinal, nervous systemcancer, nervous system lymphoma, central nervous system cancer, centralnervous system lymphoma, cervical cancer, childhood cancers, chroniclymphocytic leukemia, chronic myelogenous leukemia, chronicmyeloproliferative disorders, colon cancer, colorectal cancer, cutaneousT-cell lymphoma, lymphoid neoplasm, mycosis fungoides, Seziary Syndrome,endometrial cancer, esophageal cancer, extracranial germ cell tumor,extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer,intraocular melanoma, retinoblastoma, gallbladder cancer, gastric(stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinalstromal tumor (GIST), germ cell tumor, ovarian germ cell tumor,gestational trophoblastic tumor glioma, head and neck cancer,hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer,intraocular melanoma, ocular cancer, islet cell tumors (endocrinepancreas), Kaposi Sarcoma, kidney cancer, renal cancer, kidney cancer,laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia,chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cellleukemia, lip and oral cavity cancer, liver cancer, lung cancer,non-small cell lung cancer, small cell lung cancer, AIDS-relatedlymphoma, non-Hodgkin lymphoma, primary central nervous system lymphoma,Waldenstram macroglobulinemia, medulloblastoma, melanoma, intraocular(eye) melanoma, merkel cell carcinoma, mesothelioma malignant,mesothelioma, metastatic squamous neck cancer, mouth cancer, cancer ofthe tongue, multiple endocrine neoplasia syndrome, mycosis fungoides,myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases,chronic myelogenous leukemia, acute myeloid leukemia, multiple myeloma,chronic myeloproliferative disorders, nasopharyngeal cancer,neuroblastoma, oral cancer, oral cavity cancer, oropharyngeal cancer,ovarian cancer, ovarian epithelial cancer, ovarian low malignantpotential tumor, pancreatic cancer, islet cell pancreatic cancer,paranasal sinus and nasal cavity cancer, parathyroid cancer, penilecancer, pharyngeal cancer, pheochromocytoma, pineoblastoma andsupratentorial primitive neuroectodermal tumors, pituitary tumor, plasmacell neoplasm/multiple myeloma, pleuropulmonary blastoma, prostatecancer, rectal cancer, renal pelvis and ureter, transitional cellcancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, ewingfamily of sarcoma tumors, Kaposi Sarcoma, soft tissue sarcoma, uterinecancer, uterine sarcoma, skin cancer (non-melanoma), skin cancer(melanoma), merkel cell skin carcinoma, small intestine cancer, softtissue sarcoma, squamous cell carcinoma, stomach (gastric) cancer,supratentorial primitive neuroectodermal tumors, testicular cancer,throat cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer,transitional cell cancer of the renal pelvis and ureter and otherurinary organs, gestational trophoblastic tumor, urethral cancer,endometrial uterine cancer, uterine sarcoma, uterine corpus cancer,vaginal cancer, vulvar cancer, and Wilm's Tumor.

A “cell proliferative disorder of the hematologic system” is a cellproliferative disorder involving cells of the hematologic system. A cellproliferative disorder of the hematologic system can include lymphoma,leukemia, myeloid neoplasms, mast cell neoplasms, myelodysplasia, benignmonoclonal gammopathy, lymphomatoid granulomatosis, lymphomatoidpapulosis, polycythemia vera, chronic myelocytic leukemia, agnogenicmyeloid metaplasia, and essential thrombocythemia. A cell proliferativedisorder of the hematologic system can include hyperplasia, dysplasia,and metaplasia of cells of the hematologic system. In one aspect,compositions of the present invention may be used to treat a cancerselected from the group consisting of a hematologic cancer of thepresent invention or a hematologic cell proliferative disorder of thepresent invention, or used to identify suitable candidates for suchpurposes. A hematologic cancer of the present invention can includemultiple myeloma, lymphoma (including Hodgkin's lymphoma, non-Hodgkin'slymphoma, childhood lymphomas, and lymphomas of lymphocytic andcutaneous origin), leukemia (including childhood leukemia, hairy-cellleukemia, acute lymphocytic leukemia, acute myelocytic leukemia, chroniclymphocytic leukemia, chronic myelocytic leukemia, chronic myelogenousleukemia, and mast cell leukemia), myeloid neoplasms and mast cellneoplasms.

A “cell proliferative disorder of the lung” is a cell proliferativedisorder involving cells of the lung. Cell proliferative disorders ofthe lung can include all forms of cell proliferative disorders affectinglung cells. Cell proliferative disorders of the lung can include lungcancer, a precancer or precancerous condition of the lung, benigngrowths or lesions of the lung, and malignant growths or lesions of thelung, and metastatic lesions in tissue and organs in the body other thanthe lung. In one aspect, compositions of the present invention may beused to treat lung cancer or cell proliferative disorders of the lung,or used to identify suitable candidates for such purposes. Lung cancercan include all forms of cancer of the lung. Lung cancer can includemalignant lung neoplasms, carcinoma in situ, typical carcinoid tumors,and atypical carcinoid tumors. Lung cancer can include small cell lungcancer (“SCLC”), non-small cell lung cancer (“NSCLC”), squamous cellcarcinoma, adenocarcinoma, small cell carcinoma, large cell carcinoma,adenosquamous cell carcinoma, and mesothelioma. Lung cancer can include“scar carcinoma,” bronchioalveolar carcinoma, giant cell carcinoma,spindle cell carcinoma, and large cell neuroendocrine carcinoma. Lungcancer can include lung neoplasms having histologic and ultrastructualheterogeneity (e.g., mixed cell types).

Cell proliferative disorders of the lung can include all forms of cellproliferative disorders affecting lung cells. Cell proliferativedisorders of the lung can include lung cancer, precancerous conditionsof the lung. Cell proliferative disorders of the lung can includehyperplasia, metaplasia, and dysplasia of the lung. Cell proliferativedisorders of the lung can include asbestos-induced hyperplasia, squamousmetaplasia, and benign reactive mesothelial metaplasia. Cellproliferative disorders of the lung can include replacement of columnarepithelium with stratified squamous epithelium, and mucosal dysplasia.Individuals exposed to inhaled injurious environmental agents such ascigarette smoke and asbestos may be at increased risk for developingcell proliferative disorders of the lung. Prior lung diseases that maypredispose individuals to development of cell proliferative disorders ofthe lung can include chronic interstitial lung disease, necrotizingpulmonary disease, scleroderma, rheumatoid disease, sarcoidosis,interstitial pneumonitis, tuberculosis, repeated pneumonias, idiopathicpulmonary fibrosis, granulomata, asbestosis, fibrosing alveolitis, andHodgkin's disease.

A “cell proliferative disorder of the colon” is a cell proliferativedisorder involving cells of the colon. Preferably, the cellproliferative disorder of the colon is colon cancer. In one aspect,compositions of the present invention may be used to treat colon canceror cell proliferative disorders of the colon, or used to identifysuitable candidates for such purposes. Colon cancer can include allforms of cancer of the colon. Colon cancer can include sporadic andhereditary colon cancers. Colon cancer can include malignant colonneoplasms, carcinoma in situ, typical carcinoid tumors, and atypicalcarcinoid tumors. Colon cancer can include adenocarcinoma, squamous cellcarcinoma, and adenosquamous cell carcinoma. Colon cancer can beassociated with a hereditary syndrome selected from the group consistingof hereditary nonpolyposis colorectal cancer, familial adenomatouspolyposis, Gardner's syndrome, Peutz-Jeghers syndrome, Turcot's syndromeand juvenile polyposis. Colon cancer can be caused by a hereditarysyndrome selected from the group consisting of hereditary nonpolyposiscolorectal cancer, familial adenomatous polyposis, Gardner's syndrome,Peutz-Jeghers syndrome, Turcot's syndrome and juvenile polyposis.

Cell proliferative disorders of the colon can include all forms of cellproliferative disorders affecting colon cells. Cell proliferativedisorders of the colon can include colon cancer, precancerous conditionsof the colon, adenomatous polyps of the colon and metachronous lesionsof the colon. A cell proliferative disorder of the colon can includeadenoma. Cell proliferative disorders of the colon can be characterizedby hyperplasia, metaplasia, and dysplasia of the colon. Prior colondiseases that may predispose individuals to development of cellproliferative disorders of the colon can include prior colon cancer.Current disease that may predispose individuals to development of cellproliferative disorders of the colon can include Crohn's disease andulcerative colitis. A cell proliferative disorder of the colon can beassociated with a mutation in a gene selected from the group consistingof p53, ras, FAP and DCC. An individual can have an elevated risk ofdeveloping a cell proliferative disorder of the colon due to thepresence of a mutation in a gene selected from the group consisting ofp53, ras, FAP and DCC.

A “cell proliferative disorder of the pancreas” is a cell proliferativedisorder involving cells of the pancreas. Cell proliferative disordersof the pancreas can include all forms of cell proliferative disordersaffecting pancreatic cells. Cell proliferative disorders of the pancreascan include pancreas cancer, a precancer or precancerous condition ofthe pancreas, hyperplasia of the pancreas, and dysaplasia of thepancreas, benign growths or lesions of the pancreas, and malignantgrowths or lesions of the pancreas, and metastatic lesions in tissue andorgans in the body other than the pancreas. Pancreatic cancer includesall forms of cancer of the pancreas. Pancreatic cancer can includeductal adenocarcinoma, adenosquamous carcinoma, pleomorphic giant cellcarcinoma, mucinous adenocarcinoma, osteoclast-like giant cellcarcinoma, mucinous cystadenocarcinoma, acinar carcinoma, unclassifiedlarge cell carcinoma, small cell carcinoma, pancreatoblastoma, papillaryneoplasm, mucinous cystadenoma, papillary cystic neoplasm, and serouscystadenoma. Pancreatic cancer can also include pancreatic neoplasmshaving histologic and ultrastructual heterogeneity (e.g., mixed celltypes).

A “cell proliferative disorder of the prostate” is a cell proliferativedisorder involving cells of the prostate. Cell proliferative disordersof the prostate can include all forms of cell proliferative disordersaffecting prostate cells. Cell proliferative disorders of the prostatecan include prostate cancer, a precancer or precancerous condition ofthe prostate, benign growths or lesions of the prostate, and malignantgrowths or lesions of the prostate, and metastatic lesions in tissue andorgans in the body other than the prostate. Cell proliferative disordersof the prostate can include hyperplasia, metaplasia, and dysplasia ofthe prostate.

A “cell proliferative disorder of the skin” is a cell proliferativedisorder involving cells of the skin. Cell proliferative disorders ofthe skin can include all forms of cell proliferative disorders affectingskin cells. Cell proliferative disorders of the skin can include aprecancer or precancerous condition of the skin, benign growths orlesions of the skin, melanoma, malignant melanoma and other malignantgrowths or lesions of the skin, and metastatic lesions in tissue andorgans in the body other than the skin. Cell proliferative disorders ofthe skin can include hyperplasia, metaplasia, and dysplasia of the skin.

A “cell proliferative disorder of the ovary” is a cell proliferativedisorder involving cells of the ovary. Cell proliferative disorders ofthe ovary can include all forms of cell proliferative disordersaffecting cells of the ovary. Cell proliferative disorders of the ovarycan include a precancer or precancerous condition of the ovary, benigngrowths or lesions of the ovary, ovarian cancer, malignant growths orlesions of the ovary, and metastatic lesions in tissue and organs in thebody other than the ovary. Cell proliferative disorders of the skin caninclude hyperplasia, metaplasia, and dysplasia of cells of the ovary.

A “cell proliferative disorder of the breast” is a cell proliferativedisorder involving cells of the breast. Cell proliferative disorders ofthe breast can include all forms of cell proliferative disordersaffecting breast cells. Cell proliferative disorders of the breast caninclude breast cancer, a precancer or precancerous condition of thebreast, benign growths or lesions of the breast, and malignant growthsor lesions of the breast, and metastatic lesions in tissue and organs inthe body other than the breast. Cell proliferative disorders of thebreast can include hyperplasia, metaplasia, and dysplasia of the breast.

A cell proliferative disorder of the breast can be a precancerouscondition of the breast. Compositions of the present invention may beused to treat a precancerous condition of the breast. A precancerouscondition of the breast can include atypical hyperplasia of the breast,ductal carcinoma in situ (DCIS), intraductal carcinoma, lobularcarcinoma in situ (LCIS), lobular neoplasia, and stage 0 or grade 0growth or lesion of the breast (e.g., stage 0 or grade 0 breast cancer,or carcinoma in situ). A precancerous condition of the breast can bestaged according to the TNM classification scheme as accepted by theAmerican Joint Committee on Cancer (AJCC), where the primary tumor (T)has been assigned a stage of T0 or Tis; and where the regional lymphnodes (N) have been assigned a stage of N0; and where distant metastasis(M) has been assigned a stage of M0.

The cell proliferative disorder of the breast can be breast cancer. Inone aspect, compositions of the present invention may be used to treatbreast cancer, or used to identify suitable candidates for suchpurposes. Breast cancer may include all forms of cancer of the breast.Breast cancer can include primary epithelial breast cancers. Breastcancer can include cancers in which the breast is involved by othertumors such as lymphoma, sarcoma or melanoma. Breast cancer can includecarcinoma of the breast, ductal carcinoma of the breast, lobularcarcinoma of the breast, undifferentiated carcinoma of the breast,cystosarcoma phyllodes of the breast, angiosarcoma of the breast, andprimary lymphoma of the breast. Breast cancer can include Stage I, II,IIIA, IIIB, IIIC and IV breast cancer. Ductal carcinoma of the breastcan include invasive carcinoma, invasive carcinoma in situ withpredominant intraductal component, inflammatory breast cancer, and aductal carcinoma of the breast with a histologic type selected from thegroup consisting of comedo, mucinous (colloid), medullary, medullarywith lymphcytic infiltrate, papillary, scirrhous, and tubular. Lobularcarcinoma of the breast can include invasive lobular carcinoma withpredominant in situ component, invasive lobular carcinoma, andinfiltrating lobular carcinoma. Breast cancer can include Paget'sdisease, Paget's disease with intraductal carcinoma, and Paget's diseasewith invasive ductal carcinoma. Breast cancer can include breastneoplasms having histologic and ultrastructual heterogeneity (e.g.,mixed cell types).

Compounds of the present invention can be used to treat breast cancer,or used to identify suitable candidates for such purposes. A breastcancer that is to be treated can include familial breast cancer. Abreast cancer that is to be treated can include sporadic breast cancer.A breast cancer that is to be treated can arise in a male subject. Abreast cancer that is to be treated can arise in a female subject. Abreast cancer that is to be treated can arise in a premenopausal femalesubject or a postmenopausal female subject. A breast cancer that is tobe treated can arise in a subject equal to or older than 30 years old,or a subject younger than 30 years old. A breast cancer that is to betreated has arisen in a subject equal to or older than 50 years old, ora subject younger than 50 years old. A breast cancer that is to betreated can arise in a subject equal to or older than 70 years old, or asubject younger than 70 years old.

A breast cancer that is to be treated can be typed to identify afamilial or spontaneous mutation in BRCA1, BRCA2, or p53. A breastcancer that is to be treated can be typed as having a HER2/neu geneamplification, as overexpressing HER2/neu, or as having a low,intermediate or high level of HER2/neu expression. A breast cancer thatis to be treated can be typed for a marker selected from the groupconsisting of estrogen receptor (ER), progesterone receptor (PR), humanepidermal growth factor receptor-2, Ki-67, CA15-3, CA 27-29, and c-Met.A breast cancer that is to be treated can be typed as ER-unknown,ER-rich or ER-poor. A breast cancer that is to be treated can be typedas ER-negative or ER-positive. ER-typing of a breast cancer may beperformed by any reproducible means. ER-typing of a breast cancer may beperformed as set forth in Onkologie 27: 175-179 (2004). A breast cancerthat is to be treated can be typed as PR-unknown, PR-rich, or PR-poor. Abreast cancer that is to be treated can be typed as PR-negative orPR-positive. A breast cancer that is to be treated can be typed asreceptor positive or receptor negative. A breast cancer that is to betreated can be typed as being associated with elevated blood levels ofCA 15-3, or CA 27-29, or both.

A breast cancer that is to be treated can include a localized tumor ofthe breast. A breast cancer that is to be treated can include a tumor ofthe breast that is associated with a negative sentinel lymph node (SLN)biopsy. A breast cancer that is to be treated can include a tumor of thebreast that is associated with a positive sentinel lymph node (SLN)biopsy. A breast cancer that is to be treated can include a tumor of thebreast that is associated with one or more positive axillary lymphnodes, where the axillary lymph nodes have been staged by any applicablemethod. A breast cancer that is to be treated can include a tumor of thebreast that has been typed as having nodal negative status (e.g.,node-negative) or nodal positive status (e.g., node-positive). A breastcancer that is to be treated can include a tumor of the breast that hasmetastasized to other locations in the body. A breast cancer that is tobe treated can be classified as having metastasized to a locationselected from the group consisting of bone, lung, liver, or brain. Abreast cancer that is to be treated can be classified according to acharacteristic selected from the group consisting of metastatic,localized, regional, local-regional, locally advanced, distant,multicentric, bilateral, ipsilateral, contralateral, newly diagnosed,recurrent, and inoperable.

A compound of the present invention may be used to treat or prevent acell proliferative disorder of the breast, or to treat or prevent breastcancer, in a subject having an increased risk of developing breastcancer relative to the population at large, or used to identify suitablecandidates for such purposes. A subject with an increased risk ofdeveloping breast cancer relative to the population at large is a femalesubject with a family history or personal history of breast cancer. Asubject with an increased risk of developing breast cancer relative tothe population at large is a female subject having a germ-line orspontaneous mutation in BRCA1 or BRCA2, or both. A subject with anincreased risk of developing breast cancer relative to the population atlarge is a female subject with a family history of breast cancer and agerm-line or spontaneous mutation in BRCA1 or BRCA2, or both. A subjectwith an increased risk of developing breast cancer relative to thepopulation at large is a female who is greater than 30 years old,greater than 40 years old, greater than 50 years old, greater than 60years old, greater than 70 years old, greater than 80 years old, orgreater than 90 years old. A subject with an increased risk ofdeveloping breast cancer relative to the population at large is asubject with atypical hyperplasia of the breast, ductal carcinoma insitu (DCIS), intraductal carcinoma, lobular carcinoma in situ (LCIS),lobular neoplasia, or a stage 0 growth or lesion of the breast (e.g.,stage 0 or grade 0 breast cancer, or carcinoma in situ).

A breast cancer that is to be treated can be histologically gradedaccording to the Scarff-Bloom-Richardson system, wherein a breast tumorhas been assigned a mitosis count score of 1, 2, or 3; a nuclearpleiomorphism score of 1, 2, or 3; a tubule formation score of 1, 2, or3; and a total Scarff-Bloom-Richardson score of between 3 and 9. Abreast cancer that is to be treated can be assigned a tumor gradeaccording to the International Consensus Panel on the Treatment ofBreast Cancer selected from the group consisting of grade 1, grade 1-2,grade 2, grade 2-3, or grade 3.

In one embodiment, provided herein is a method of treating breast cancercomprising administering to a subject in need thereof an effectiveamount of the hydrobromide of Compound I.

In another embodiment, provided herein is a method of treating breastcancer comprising administering to a subject in need thereof aneffective amount of Polymorph A.

A cancer that is to be treated can be staged according to the AmericanJoint Committee on Cancer (AJCC) TNM classification system, where thetumor (T) has been assigned a stage of TX, T1, T1mic, T1a, T1b, T1c, T2,T3, T4, T4a, T4b, T4c, or T4d; and where the regional lymph nodes (N)have been assigned a stage of NX, N0, N1, N2, N2a, N2b, N3, N3a, N3b, orN3c; and where distant metastasis (M) can be assigned a stage of MX, M0,or M1. A cancer that is to be treated can be staged according to anAmerican Joint Committee on Cancer (AJCC) classification as Stage I,Stage IIA, Stage IIB, Stage IIIA, Stage IIIB, Stage IIIC, or Stage IV. Acancer that is to be treated can be assigned a grade according to anAJCC classification as Grade GX (e.g., grade cannot be assessed), Grade1, Grade 2, Grade 3 or Grade 4. A cancer that is to be treated can bestaged according to an AJCC pathologic classification (pN) of pNX, pN0,PN0 (I−), PN0 (I+), PN0 (mol−), PN0 (mol+), PN1, PN1(mi), PN1a, PN1b,PN1c, pN2, pN2a, pN2b, pN3, pN3a, pN3b, or pN3c.

A cancer that is to be treated can include a tumor that has beendetermined to be less than or equal to about 2 centimeters in diameter.A cancer that is to be treated can include a tumor that has beendetermined to be from about 2 to about 5 centimeters in diameter. Acancer that is to be treated can include a tumor that has beendetermined to be greater than or equal to about 3 centimeters indiameter. A cancer that is to be treated can include a tumor that hasbeen determined to be greater than 5 centimeters in diameter. A cancerthat is to be treated can be classified by microscopic appearance aswell differentiated, moderately differentiated, poorly differentiated,or undifferentiated. A cancer that is to be treated can be classified bymicroscopic appearance with respect to mitosis count (e.g., amount ofcell division) or nuclear pleiomorphism (e.g., change in cells). Acancer that is to be treated can be classified by microscopic appearanceas being associated with areas of necrosis (e.g., areas of dying ordegenerating cells). A cancer that is to be treated can be classified ashaving an abnormal karyotype, having an abnormal number of chromosomes,or having one or more chromosomes that are abnormal in appearance. Acancer that is to be treated can be classified as being aneuploid,triploid, tetraploid, or as having an altered ploidy. A cancer that isto be treated can be classified as having a chromosomal translocation,or a deletion or duplication of an entire chromosome, or a region ofdeletion, duplication or amplification of a portion of a chromosome.

A cancer that is to be treated can be evaluated by DNA cytometry, flowcytometry, or image cytometry. A cancer that is to be treated can betyped as having 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of cellsin the synthesis stage of cell division (e.g., in S phase of celldivision). A cancer that is to be treated can be typed as having a lowS-phase fraction or a high S-phase fraction.

As used herein, a “normal cell” is a cell that cannot be classified aspart of a “cell proliferative disorder”. A normal cell lacks unregulatedor abnormal growth, or both, that can lead to the development of anunwanted condition or disease. Preferably, a normal cell possessesnormally functioning cell cycle checkpoint control mechanisms.

As used herein, “contacting a cell” refers to a condition in which acompound or other composition of matter is in direct contact with acell, or is close enough to induce a desired biological effect in acell.

As used herein, “candidate compound” refers to a compound of the presentinvention (i.e., the hydrobromide of Compound I, as well as Polymorph A)that has been or will be tested in one or more in vitro or in vivobiological assays, in order to determine if that compound is likely toelicit a desired biological or medical response in a cell, tissue,system, animal or human that is being sought by a researcher orclinician. A candidate compound is a compound of the present invention.The biological or medical response can be the treatment of cancer. Thebiological or medical response can be treatment or prevention of a cellproliferative disorder. The biological response or effect can alsoinclude a change in cell proliferation or growth that occurs in vitro orin an animal model, as well as other biological changes that areobservable in vitro. In vitro or in vivo biological assays can include,but are not limited to, enzymatic activity assays, electrophoreticmobility shift assays, reporter gene assays, in vitro cell viabilityassays, and the assays described herein.

As used herein, “monotherapy” refers to the administration of a singleactive or therapeutic compound to a subject in need thereof. Preferably,monotherapy will involve administration of a therapeutically effectiveamount of an active compound. For example, cancer monotherapy with oneof the compound of the present invention (i.e., the hydrobromide ofCompound I, as well as Polymorph A) to a subject in need of treatment ofcancer. Monotherapy may be contrasted with combination therapy, in whicha combination of multiple active compounds is administered, preferablywith each component of the combination present in a therapeuticallyeffective amount. In one aspect, monotherapy with a compound of thepresent invention is more effective than combination therapy in inducinga desired biological effect.

As used herein, “treating” or “treat” describes the management and careof a patient for the purpose of combating a disease, condition, ordisorder and includes the administration of a compound of the presentinvention (i.e., the hydrobromide of Compound I, as well as Polymorph A)to alleviate the symptoms or complications of a disease, condition ordisorder, or to eliminate the disease, condition or disorder. The term“treat” can also include treatment of a cell in vitro or an animalmodel.

A compound of the present invention (i.e., the hydrobromide of CompoundI, as well as Polymorph A) can also be used to prevent a disease,condition or disorder, or used to identify suitable candidates for suchpurposes. As used herein, “preventing” or “prevent” describes reducingor eliminating the onset of the symptoms or complications of thedisease, condition or disorder.

As used herein, the term “alleviate” is meant to describe a process bywhich the severity of a sign or symptom of a disorder is decreased.Importantly, a sign or symptom can be alleviated without beingeliminated. In a preferred embodiment, the administration ofpharmaceutical compositions of the invention leads to the elimination ofa sign or symptom, however, elimination is not required. Effectivedosages are expected to decrease the severity of a sign or symptom. Forinstance, a sign or symptom of a disorder such as cancer, which canoccur in multiple locations, is alleviated if the severity of the canceris decreased within at least one of multiple locations.

As used herein, the term “severity” is meant to describe the potentialof cancer to transform from a precancerous, or benign, state into amalignant state. Alternatively, or in addition, severity is meant todescribe a cancer stage, for example, according to the TNM system(accepted by the International Union Against Cancer (UICC) and theAmerican Joint Committee on Cancer (AJCC)) or by other art-recognizedmethods. Cancer stage refers to the extent or severity of the cancer,based on factors such as the location of the primary tumor, tumor size,number of tumors, and lymph node involvement (spread of cancer intolymph nodes). Alternatively, or in addition, severity is meant todescribe the tumor grade by art-recognized methods (see, National CancerInstitute, www.cancer.gov). Tumor grade is a system used to classifycancer cells in terms of how abnormal they look under a microscope andhow quickly the tumor is likely to grow and spread. Many factors areconsidered when determining tumor grade, including the structure andgrowth pattern of the cells. The specific factors used to determinetumor grade vary with each type of cancer. Severity also describes ahistologic grade, also called differentiation, which refers to how muchthe tumor cells resemble normal cells of the same tissue type (see,National Cancer Institute, www.cancer.gov). Furthermore, severitydescribes a nuclear grade, which refers to the size and shape of thenucleus in tumor cells and the percentage of tumor cells that aredividing (see, National Cancer Institute, www.cancer.gov).

In another aspect of the invention, severity describes the degree towhich a tumor has secreted growth factors, degraded the extracellularmatrix, become vascularized, lost adhesion to juxtaposed tissues, ormetastasized. Moreover, severity describes the number of locations towhich a primary tumor has metastasized. Finally, severity includes thedifficulty of treating tumors of varying types and locations. Forexample, inoperable tumors, those cancers which have greater access tomultiple body systems (hematological and immunological tumors), andthose which are the most resistant to traditional treatments areconsidered most severe. In these situations, prolonging the lifeexpectancy of the subject and/or reducing pain, decreasing theproportion of cancerous cells or restricting cells to one system, andimproving cancer stage/tumor grade/histological grade/nuclear grade areconsidered alleviating a sign or symptom of the cancer.

As used herein the term “symptom” is defined as an indication ofdisease, illness, injury, or that something is not right in the body.Symptoms are felt or noticed by the individual experiencing the symptom,but may not easily be noticed by others. Others are defined asnon-health-care professionals.

As used herein the term “sign” is also defined as an indication thatsomething is not right in the body. But signs are defined as things thatcan be seen by a doctor, nurse, or other health care professional.

Cancer is a group of diseases that may cause almost any sign or symptom.The signs and symptoms will depend on where the cancer is, the size ofthe cancer, and how much it affects the nearby organs or structures. Ifa cancer spreads (metastasizes), then symptoms may appear in differentparts of the body.

As a cancer grows, it begins to push on nearby organs, blood vessels,and nerves. This pressure creates some of the signs and symptoms ofcancer. If the cancer is in a critical area, such as certain parts ofthe brain, even the smallest tumor can cause early symptoms.

But sometimes cancers start in places where it does not cause anysymptoms until the cancer has grown quite large. Pancreas cancers, forexample, do not usually grow large enough to be felt from the outside ofthe body. Some pancreatic cancers do not cause symptoms until they beginto grow around nearby nerves (this causes a backache). Others growaround the bile duct, which blocks the flow of bile and leads to ayellowing of the skin known as jaundice. By the time a pancreatic cancercauses these signs or symptoms, it has usually reached an advancedstage.

A cancer may also cause symptoms such as fever, fatigue, or weight loss.This may be because cancer cells use up much of the body's energy supplyor release substances that change the body's metabolism. Or the cancermay cause the immune system to react in ways that produce thesesymptoms.

Sometimes, cancer cells release substances into the bloodstream thatcause symptoms not usually thought to result from cancers. For example,some cancers of the pancreas can release substances which cause bloodclots to develop in veins of the legs. Some lung cancers makehormone-like substances that affect blood calcium levels, affectingnerves and muscles and causing weakness and dizziness

Cancer presents several general signs or symptoms that occur when avariety of subtypes of cancer cells are present. Most people with cancerwill lose weight at some time with their disease. An unexplained(unintentional) weight loss of 10 pounds or more may be the first signof cancer, particularly cancers of the pancreas, stomach, esophagus, orlung.

Fever is very common with cancer, but is more often seen in advanceddisease. Almost all patients with cancer will have fever at some time,especially if the cancer or its treatment affects the immune system andmakes it harder for the body to fight infection. Less often, fever maybe an early sign of cancer, such as with leukemia or lymphoma.

Fatigue may be an important symptom as cancer progresses. It may happenearly, though, in cancers such as with leukemia, or if the cancer iscausing an ongoing loss of blood, as in some colon or stomach cancers.

Pain may be an early symptom with some cancers such as bone cancers ortesticular cancer. But most often pain is a symptom of advanced disease.

Along with cancers of the skin (see next section), some internal cancerscan cause skin signs that can be seen. These changes include the skinlooking darker (hyperpigmentation), yellow (jaundice), or red(erythema); itching; or excessive hair growth.

Alternatively, or in addition, cancer subtypes present specific signs orsymptoms. Changes in bowel habits or bladder function could indicatecancer. Long-term constipation, diarrhea, or a change in the size of thestool may be a sign of colon cancer. Pain with urination, blood in theurine, or a change in bladder function (such as more frequent or lessfrequent urination) could be related to bladder or prostate cancer.

Changes in skin condition or appearance of a new skin condition couldindicate cancer. Skin cancers may bleed and look like sores that do notheal. A long-lasting sore in the mouth could be an oral cancer,especially in patients who smoke, chew tobacco, or frequently drinkalcohol. Sores on the penis or vagina may either be signs of infectionor an early cancer.

Unusual bleeding or discharge could indicate cancer. Unusual bleedingcan happen in either early or advanced cancer. Blood in the sputum(phlegm) may be a sign of lung cancer. Blood in the stool (or a dark orblack stool) could be a sign of colon or rectal cancer. Cancer of thecervix or the endometrium (lining of the uterus) can cause vaginalbleeding. Blood in the urine may be a sign of bladder or kidney cancer.A bloody discharge from the nipple may be a sign of breast cancer.

A thickening or lump in the breast or in other parts of the body couldindicate the presence of a cancer. Many cancers can be felt through theskin, mostly in the breast, testicle, lymph nodes (glands), and the softtissues of the body. A lump or thickening may be an early or late signof cancer. Any lump or thickening could be indicative of cancer,especially if the formation is new or has grown in size.

Indigestion or trouble swallowing could indicate cancer. While thesesymptoms commonly have other causes, indigestion or swallowing problemsmay be a sign of cancer of the esophagus, stomach, or pharynx (throat).

Recent changes in a wart or mole could be indicative of cancer. Anywart, mole, or freckle that changes in color, size, or shape, or losesits definite borders indicates the potential development of cancer. Forexample, the skin lesion may be a melanoma.

A persistent cough or hoarseness could be indicative of cancer. A coughthat does not go away may be a sign of lung cancer. Hoarseness can be asign of cancer of the larynx (voice box) or thyroid.

While the signs and symptoms listed above are the more common ones seenwith cancer, there are many others that are less common and are notlisted here. However, all art-recognized signs and symptoms of cancerare contemplated and encompassed by the instant invention.

Treating cancer can result in a reduction in size of a tumor. Areduction in size of a tumor may also be referred to as “tumorregression”. Preferably, after treatment, tumor size is reduced by 5% orgreater relative to its size prior to treatment; more preferably, tumorsize is reduced by 10% or greater; more preferably, reduced by 20% orgreater; more preferably, reduced by 30% or greater; more preferably,reduced by 40% or greater; even more preferably, reduced by 50% orgreater; and most preferably, reduced by greater than 75% or greater.Size of a tumor may be measured by any reproducible means ofmeasurement. The size of a tumor may be measured as a diameter of thetumor.

Treating cancer can result in a reduction in tumor volume. Preferably,after treatment, tumor volume is reduced by 5% or greater relative toits size prior to treatment; more preferably, tumor volume is reduced by10% or greater; more preferably, reduced by 20% or greater; morepreferably, reduced by 30% or greater; more preferably, reduced by 40%or greater; even more preferably, reduced by 50% or greater; and mostpreferably, reduced by greater than 75% or greater. Tumor volume may bemeasured by any reproducible means of measurement.

Treating cancer results in a decrease in number of tumors. Preferably,after treatment, tumor number is reduced by 5% or greater relative tonumber prior to treatment; more preferably, tumor number is reduced by10% or greater; more preferably, reduced by 20% or greater; morepreferably, reduced by 30% or greater; more preferably, reduced by 40%or greater; even more preferably, reduced by 50% or greater; and mostpreferably, reduced by greater than 75%. Number of tumors may bemeasured by any reproducible means of measurement. The number of tumorsmay be measured by counting tumors visible to the naked eye or at aspecified magnification. Preferably, the specified magnification is 2×,3×, 4×, 5×, 10×, or 50×.

Treating cancer can result in a decrease in number of metastatic lesionsin other tissues or organs distant from the primary tumor site.Preferably, after treatment, the number of metastatic lesions is reducedby 5% or greater relative to number prior to treatment; more preferably,the number of metastatic lesions is reduced by 10% or greater; morepreferably, reduced by 20% or greater; more preferably, reduced by 30%or greater; more preferably, reduced by 40% or greater; even morepreferably, reduced by 50% or greater; and most preferably, reduced bygreater than 75%. The number of metastatic lesions may be measured byany reproducible means of measurement. The number of metastatic lesionsmay be measured by counting metastatic lesions visible to the naked eyeor at a specified magnification. Preferably, the specified magnificationis 2×, 3×, 4×, 5×, 10×, or 50×.

Treating cancer can result in an increase in average survival time of apopulation of treated subjects in comparison to a population receivingcarrier alone. Preferably, the average survival time is increased bymore than 30 days; more preferably, by more than 60 days; morepreferably, by more than 90 days; and most preferably, by more than 120days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active compound. An increase in average survival timeof a population may also be measured, for example, by calculating for apopulation the average length of survival following completion of afirst round of treatment with an active compound.

Treating cancer can result in an increase in average survival time of apopulation of treated subjects in comparison to a population ofuntreated subjects. Preferably, the average survival time is increasedby more than 30 days; more preferably, by more than 60 days; morepreferably, by more than 90 days; and most preferably, by more than 120days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active compound. An increase in average survival timeof a population may also be measured, for example, by calculating for apopulation the average length of survival following completion of afirst round of treatment with an active compound.

Treating cancer can result in increase in average survival time of apopulation of treated subjects in comparison to a population receivingmonotherapy with a drug that is not a compound of the present invention.Preferably, the average survival time is increased by more than 30 days;more preferably, by more than 60 days; more preferably, by more than 90days; and most preferably, by more than 120 days. An increase in averagesurvival time of a population may be measured by any reproducible means.An increase in average survival time of a population may be measured,for example, by calculating for a population the average length ofsurvival following initiation of treatment with an active compound. Anincrease in average survival time of a population may also be measured,for example, by calculating for a population the average length ofsurvival following completion of a first round of treatment with anactive compound.

Treating cancer can result in a decrease in the mortality rate of apopulation of treated subjects in comparison to a population receivingcarrier alone. Treating cancer can result in a decrease in the mortalityrate of a population of treated subjects in comparison to an untreatedpopulation. Treating cancer can result in a decrease in the mortalityrate of a population of treated subjects in comparison to a populationreceiving monotherapy with a drug that is not a compound of the presentinvention. Preferably, the mortality rate is decreased by more than 2%;more preferably, by more than 5%; more preferably, by more than 10%; andmost preferably, by more than 25%. A decrease in the mortality rate of apopulation of treated subjects may be measured by any reproduciblemeans. A decrease in the mortality rate of a population may be measured,for example, by calculating for a population the average number ofdisease-related deaths per unit time following initiation of treatmentwith an active compound. A decrease in the mortality rate of apopulation may also be measured, for example, by calculating for apopulation the average number of disease-related deaths per unit timefollowing completion of a first round of treatment with an activecompound.

Treating cancer can result in a decrease in tumor growth rate.Preferably, after treatment, tumor growth rate is reduced by at least 5%relative to number prior to treatment; more preferably, tumor growthrate is reduced by at least 10%; more preferably, reduced by at least20%; more preferably, reduced by at least 30%; more preferably, reducedby at least 40%; more preferably, reduced by at least 50%; even morepreferably, reduced by at least 50%; and most preferably, reduced by atleast 75%. Tumor growth rate may be measured by any reproducible meansof measurement. Tumor growth rate can be measured according to a changein tumor diameter per unit time.

Treating cancer can result in a decrease in tumor regrowth. Preferably,after treatment, tumor regrowth is less than 5%; more preferably, tumorregrowth is less than 10%; more preferably, less than 20%; morepreferably, less than 30%; more preferably, less than 40%; morepreferably, less than 50%; even more preferably, less than 50%; and mostpreferably, less than 75%. Tumor regrowth may be measured by anyreproducible means of measurement. Tumor regrowth is measured, forexample, by measuring an increase in the diameter of a tumor after aprior tumor shrinkage that followed treatment. A decrease in tumorregrowth is indicated by failure of tumors to reoccur after treatmenthas stopped.

Treating or preventing a cell proliferative disorder can result in areduction in the rate of cellular proliferation. Preferably, aftertreatment, the rate of cellular proliferation is reduced by at least 5%;more preferably, by at least 10%; more preferably, by at least 20%; morepreferably, by at least 30%; more preferably, by at least 40%; morepreferably, by at least 50%; even more preferably, by at least 50%; andmost preferably, by at least 75%. The rate of cellular proliferation maybe measured by any reproducible means of measurement. The rate ofcellular proliferation is measured, for example, by measuring the numberof dividing cells in a tissue sample per unit time.

Treating or preventing a cell proliferative disorder can result in areduction in the proportion of proliferating cells. Preferably, aftertreatment, the proportion of proliferating cells is reduced by at least5%; more preferably, by at least 10%; more preferably, by at least 20%;more preferably, by at least 30%; more preferably, by at least 40%; morepreferably, by at least 50%; even more preferably, by at least 50%; andmost preferably, by at least 75%. The proportion of proliferating cellsmay be measured by any reproducible means of measurement. Preferably,the proportion of proliferating cells is measured, for example, byquantifying the number of dividing cells relative to the number ofnondividing cells in a tissue sample. The proportion of proliferatingcells can be equivalent to the mitotic index.

Treating or preventing a cell proliferative disorder can result in adecrease in size of an area or zone of cellular proliferation.Preferably, after treatment, size of an area or zone of cellularproliferation is reduced by at least 5% relative to its size prior totreatment; more preferably, reduced by at least 10%; more preferably,reduced by at least 20%; more preferably, reduced by at least 30%; morepreferably, reduced by at least 40%; more preferably, reduced by atleast 50%; even more preferably, reduced by at least 50%; and mostpreferably, reduced by at least 75%. Size of an area or zone of cellularproliferation may be measured by any reproducible means of measurement.The size of an area or zone of cellular proliferation may be measured asa diameter or width of an area or zone of cellular proliferation.

Treating or preventing a cell proliferative disorder can result in adecrease in the number or proportion of cells having an abnormalappearance or morphology. Preferably, after treatment, the number ofcells having an abnormal morphology is reduced by at least 5% relativeto its size prior to treatment; more preferably, reduced by at least10%; more preferably, reduced by at least 20%; more preferably, reducedby at least 30%; more preferably, reduced by at least 40%; morepreferably, reduced by at least 50%; even more preferably, reduced by atleast 50%; and most preferably, reduced by at least 75%. An abnormalcellular appearance or morphology may be measured by any reproduciblemeans of measurement. An abnormal cellular morphology can be measured bymicroscopy, e.g., using an inverted tissue culture microscope. Anabnormal cellular morphology can take the form of nuclear pleiomorphism.

As used herein, the term “selectively” means tending to occur at ahigher frequency in one population than in another population. Thecompared populations can be cell populations. Preferably, a compound ofthe present invention (i.e., the hydrobromide of Compound I, as well asPolymorph A) acts selectively on a cancer or precancerous cell but noton a normal cell. Preferably, a compound of the present invention actsselectively to modulate one molecular target (e.g., a target proteinmethyltransferase) but does not significantly modulate another moleculartarget (e.g., a non-target protein methyltransferase). The inventionalso provides a method for selectively inhibiting the activity of anenzyme, such as a protein methyltransferase. Preferably, an event occursselectively in population A relative to population B if it occursgreater than two times more frequently in population A as compared topopulation B. An event occurs selectively if it occurs greater than fivetimes more frequently in population A. An event occurs selectively if itoccurs greater than ten times more frequently in population A; morepreferably, greater than fifty times; even more preferably, greater than100 times; and most preferably, greater than 1000 times more frequentlyin population A as compared to population B. For example, cell deathwould be said to occur selectively in cancer cells if it occurredgreater than twice as frequently in cancer cells as compared to normalcells.

A compound of the present invention can modulate the activity of amolecular target (e.g., a target protein methyltransferase). Modulatingrefers to stimulating or inhibiting an activity of a molecular target.Preferably, a compound of the present invention modulates the activityof a molecular target if it stimulates or inhibits the activity of themolecular target by at least 2-fold relative to the activity of themolecular target under the same conditions but lacking only the presenceof said compound. More preferably, a compound of the present inventionmodulates the activity of a molecular target if it stimulates orinhibits the activity of the molecular target by at least 5-fold, atleast 10-fold, at least 20-fold, at least 50-fold, at least 100-foldrelative to the activity of the molecular target under the sameconditions but lacking only the presence of said compound. The activityof a molecular target may be measured by any reproducible means. Theactivity of a molecular target may be measured in vitro or in vivo. Forexample, the activity of a molecular target may be measured in vitro byan enzymatic activity assay or a DNA binding assay, or the activity of amolecular target may be measured in vivo by assaying for expression of areporter gene.

A compound of the present invention (i.e., the hydrobromide of CompoundI, as well as Polymorph A) does not significantly modulate the activityof a molecular target if the addition of the compound does not stimulateor inhibit the activity of the molecular target by greater than 10%relative to the activity of the molecular target under the sameconditions but lacking only the presence of said compound.

As used herein, the term “isozyme selective” means preferentialinhibition or stimulation of a first isoform of an enzyme in comparisonto a second isoform of an enzyme (e.g., preferential inhibition orstimulation of a protein methyltransferase isozyme alpha in comparisonto a protein methyltransferase isozyme beta). Preferably, a compound ofthe present invention demonstrates a minimum of a fourfold differential,preferably a tenfold differential, more preferably a fifty folddifferential, in the dosage required to achieve a biological effect.Preferably, a compound of the present invention demonstrates thisdifferential across the range of inhibition, and the differential isexemplified at the IC₅₀, i.e., a 50% inhibition, for a molecular targetof interest.

Administering a compound of the present invention to a cell or a subjectin need thereof can result in modulation (i.e., stimulation orinhibition) of an activity of a protein methyltransferase of interest.

Treating cancer or a cell proliferative disorder can result in celldeath, and preferably, cell death results in a decrease of at least 10%in number of cells in a population. More preferably, cell death means adecrease of at least 20%; more preferably, a decrease of at least 30%;more preferably, a decrease of at least 40%; more preferably, a decreaseof at least 50%; most preferably, a decrease of at least 75%. Number ofcells in a population may be measured by any reproducible means. Anumber of cells in a population can be measured by fluorescenceactivated cell sorting (FACS), immunofluorescence microscopy and lightmicroscopy. Methods of measuring cell death are as shown in Li et al.,Proc Natl Acad Sci USA. 100(5): 2674-8, 2003. In an aspect, cell deathoccurs by apoptosis.

Preferably, an effective amount of a compound of the present inventionis not significantly cytotoxic to normal cells. A therapeuticallyeffective amount of a compound is not significantly cytotoxic to normalcells if administration of the compound in a therapeutically effectiveamount does not induce cell death in greater than 10% of normal cells. Atherapeutically effective amount of a compound does not significantlyaffect the viability of normal cells if administration of the compoundin a therapeutically effective amount does not induce cell death ingreater than 10% of normal cells. In an aspect, cell death occurs byapoptosis.

Contacting a cell with a compound of the present invention can induce oractivate cell death selectively in cancer cells. Administering to asubject in need thereof a compound of the present invention can induceor activate cell death selectively in cancer cells. Contacting a cellwith a compound of the present invention can induce cell deathselectively in one or more cells affected by a cell proliferativedisorder. Preferably, administering to a subject in need thereof acompound of the present invention induces cell death selectively in oneor more cells affected by a cell proliferative disorder.

The present invention relates to a method of treating or preventingcancer (e.g, the course of which can be influenced by modulatingEZH2-mediated protein methylation) by administering a compound of thepresent invention (i.e., the hydrobromide of Compound I, as well asPolymorph A) to a subject in need thereof, where administration of thecompound of the present invention results in one or more of thefollowing: prevention of cancer cell proliferation by accumulation ofcells in one or more phases of the cell cycle (e.g. G1, G1/S, G2/M), orinduction of cell senescence, or promotion of tumor celldifferentiation; promotion of cell death in cancer cells viacytotoxicity, necrosis or apoptosis, without a significant amount ofcell death in normal cells, antitumor activity in animals with atherapeutic index of at least 2. As used herein, “therapeutic index” isthe maximum tolerated dose divided by the efficacious dose. The presentinvention also relates to a method used to identify suitable candidatesfor treating or preventing cancer.

One skilled in the art may refer to general reference texts for detaileddescriptions of known techniques discussed herein or equivalenttechniques. These texts include Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Inc. (2005); Sambrook et al.,Molecular Cloning, A Laboratory Manual (3^(rd) edition), Cold SpringHarbor Press, Cold Spring Harbor, N.Y. (2000); Coligan et al., CurrentProtocols in Immunology, John Wiley & Sons, N.Y.; Enna et al., CurrentProtocols in Pharmacology, John Wiley & Sons, N.Y.; Fingl et al., ThePharmacological Basis of Therapeutics (1975), Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 18^(th) edition (1990).These texts can, of course, also be referred to in making or using anaspect of the invention.

Exemplification Materials and Methods

Powder X-Ray Diffraction

PXRD for all samples was taken on a Rigaku MultiFlex (Target: Cu; Tubevoltage: 40 kV; Tube current: 30 mA).

Differential Scanning Calorimetry

DSC for all samples was taken on a Mettler-Toledo DSC 1/700 (Runconditions: Initial temperature 35° C., Final temp 325° C., Heating rate30° C./min).

X-Ray Crystallography

A colorless plate crystal with dimensions 0.28×0.22×0.06 mm was mountedon a Nylon loop using very small amount of paratone oil. Data werecollected using a Bruker CCD (charge coupled device) baseddiffractometer equipped with an Oxford Cryostream low-temperatureapparatus operating at 173 K. Data were measured using omega and phiscans of 0.5° per frame for 45 s. The total number of images was basedon results from the program COSMO where redundancy was expected to be4.0 and completeness to100% out to 0.83 Å. Cell parameters wereretrieved using APEX II software and refined using SAINT on all observedreflections. Data reduction was performed using the SAINT software whichcorrects for Lp. Scaling and absorption corrections were applied usingSADABS multi-scan technique, supplied by George Sheldrick. Thestructures are solved by the direct method using the SHELXS-97 programand refined by least squares method on F², SHELXL-97, which areincorporated in SHELXTL-PC V 6.10.

The structure shown in FIG. 11 was solved in the space group P2₁/c(#14). All non-hydrogen atoms are refined anisotropically. Hydrogenswere calculated by geometrical methods and refined as a riding model.The crystal used for the diffraction study showed no decompositionduring data collection. All drawings are done at 50% ellipsoids.

Dynamic Vapor Sorption

DVS was measured on a VTI Model SGA-100 system. Measurement method: Therelative humidity (RH) was changed in a controlled fashion, in 5% stepsfrom 5.0% to 95.0% then back to 5.0% using the gravimetric vaporsorption system, and the weight percentage change (wt %) of the sampleat each stage was measured.

HPLC

HPLC was conducted on an Agilent 1200 HPLC quaternary pump, low pressuremixing, with an in-line degasser. Analytical method conditions: 8 μLsample (20 mg of ER-581982-06 diluted with 50 mL of a methanol toprovide approximately 0.4 mg/mL solution) was injected onto a AgilentZorbax Eclipse XDB-C18 (4.6×150 mm, 3.5 um), Chromatography conditions:mobile phase A, water with 5 mM ammonium formate; mobile phase B, 5 mMammonium formate in 50/45/5 acetonitrile/methanol/water; flow rate, 1.5ml/min.; gradient: isocratic at 10% B from 0 to 3 min; linear increaseto 70% B from 3 to 7 min; isocratic at 70% B from 7 to 12 min; linearincrease to 100% B from 12 to 15 min isocratic at 100% B from 15 to 20min; column temperature, 35° C.; detection, UV 230 nm. Approximateretention time of Compound I=10.7 min.

Synthesis of Polymorph A

5-bromo-2-methyl-3-nitrobenzoic acid stirred solution of2-methyl-3-nitrobenzoic acid (100 g, 552 mmol) in conc. H₂SO₄ (400 mL),1,3-dibromo-5,5-dimethyl-2,4-imidazolidinedione (88 g, 308 mmol) wasadded in a portion wise manner at room temperature and the reactionmixture was then stirred at room temperature for 5 h. The reactionmixture was poured onto ice cold water, the precipitated solid wasfiltered off, washed with water and dried under vacuum to afford thedesired compound as a solid (140 g, 98%). The isolated compound wastaken directly into the next step. ¹H NMR (DMSO-d₆, 400 MHz) δ 8.31 (s,1H), 8.17 (s, 1H), 2.43 (s, 3H).

Methyl 5-bromo-2-methyl-3-nitrobenzoate To a stirred solution of5-bromo-2-methyl-3-nitrobenzoic acid (285 g, 1105 mmol) in DMF (2.8 L)at room temperature was added sodium carbonate (468 g, 4415 mmol)followed by addition of methyl iodide (626.6 g, 4415 mmol). Theresulting reaction mixture was heated at 60° C. for 8 h. Aftercompletion (monitored by TLC), the reaction mixture was filtered (toremove sodium carbonate) and washed with ethyl acetate (1 L×3). Thecombined filtrate was washed with water (3 L×5) and the aqueous phasewas back extracted with ethyl acetate (1 L×3). The combined organiclayers were dried over anhydrous sodium sulfate, filtered andconcentrated under reduced pressure to afford the title compound as asolid (290 g, 97% yield). The isolated compound was taken directly intothe next step. ¹H NMR (CDCl₃, 400 MHz) δ 8.17 (s, 1H), 7.91 (s, 1H),3.96 (s, 3H), 2.59 (s, 3H).

Methyl 3-amino-5-bromo-2-methylbenzoate (1) To a stirred solution ofmethyl 5-bromo-2-methyl-3-nitrobenzoate (290 g, 1058 mmol) in ethanol(1.5 L) was added aqueous ammonium chloride (283 g, 5290 mmol dissolvedin 1.5 L water). The resulting mixture was stirred at 80° C. to whichiron powder (472 g, 8451 mmol) was added in a portion wise manner. Theresulting reaction mixture was heated at 80° C. for 12 h. Uponcompletion as determined by TLC, the reaction mixture was hot filteredover Celite® and the celite bed was washed with methanol (5 L) followedby washing with 30% MeOH in DCM (5 L). The combined filtrate wasconcentrated in-vacuo, the residue obtained was diluted with aqueoussodium bicarbonate solution (2 L) and extracted with ethyl acetate (5L×3). The combined organic layers were dried over anhydrous sodiumsulfate, filtered and concentrated under reduced pressure to afford thetitle compound as a solid (220 g, 85%). The compound was taken directlyinto the next step. ¹H NMR (CDCl₃, 400 MHz) δ 7.37 (s, 1H), 6.92 (s,1H), 3.94 (s, 3H), 3.80 (bs, 2H), 2.31 (s, 3H).

Methyl 5-bromo-2-methyl-3-((tetrahydro-2H-pyran-4-yl) amino) benzoate(2) A reactor was charged with methyl 3-amino-5-bromo-2-methylbenzoate(455.8 g, 1.87 mol), 1,2-Dichloroethane (4.56 L), and acetic acid (535ml, 9.34 mol). To the mixture were added dihydro-2H-pyran-4(3H)-one (280g, 2.80 mol) and sodium triacetoxyborohydride (594 g, 2.80 mol)maintaining the internal temperature below 40° C. The mixture wasstirred at 25° C. for 2.5 h and then the reaction was quenched with asolution of sodium hydroxide (448 g, 11.20 mol) in water (5.61 L). Afterstirring for 20 minutes at ambient temperature, the organic layer wasseparated and the aqueous layer was extracted with ethyl acetate (3.65L). The organic layers were combined, washed with brine (1.5 L), andconcentrated under vacuum.

The residue was treated with ethyl acetate (1.8 L) and heated to 65-70°C. The mixture was stirred at 65-70° C. for 15 minutes to give a clearsolution and then treated with n-heptane (7.3 L) maintaining thetemperature between 60-70° C. Once the heptane was completely added tothe solution, the mixture was held at 65-70° C. for 15 minutes and thenallowed to cool to 18-22° C. over 3 h. The resulting suspension wasstirred at 18-22° C. for 4 h, cooled to 0-5° C. over 1 h, and held at0-5° C. for 2 h. The precipitate was filtered, washed twice withn-heptane (1.4 L), and dried under vacuum to give the title compound(540 g, 88%). The XRPD pattern of this compound is shown in FIG. 17.

Methyl 5-bromo-3-(ethyl (tetrahydro-2H-pyran-4-yl)amino)-2-methylbenzoate (3) To a stirred solution of methyl5-bromo-2-methyl-3-((tetrahydro-2H-pyran-4-yl) amino) benzoate (14 g,42.7 mmol) in dichloroethane (150 mL) was added acetaldehyde (3.75 g,85.2 mmol) and acetic acid (15.3 g, 256 mmol). The resulting reactionmixture was stirred at room temperature for 15 minutes. The mixture wascooled to 0° C. and sodium triacetoxyborohydride (27 g, 128 mmol) wasadded. The reaction mixture was stirred at room temperature for 3 hours.Upon completion of the reaction as determined by TLC, aqueous sodiumbicarbonate solution was added to the reaction mixture until a pH 7-8was obtained, the organic phase was separated and the aqueous phase wasextracted with ethyl acetate. The combined organic layers were driedover anhydrous sodium sulfate, filtered and concentrated under reducedpressure. The crude compound was purified by column chromatography(100-200 mesh silica gel) eluting with ethyl acetate: hexane to affordthe desired compound as a viscous liquid (14 g, 93%). ¹H NMR (DMSO-d₆,400 MHz) δ 7.62 (s, 1H), 7.52 (s, 1H), 3.80 (bs, 5H), 3.31 (t, 2H),2.97-3.05 (m, 2H), 2.87-2.96 (m, 1H), 2.38 (s, 3H), 1.52-1.61 (m, 2H),1.37-1.50 (m, 2H), 0.87 (t, 3H, J=6.8 Hz).

Methyl5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxylate(4): A mixture of methyl5-bromo-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methylbenzoate (580g, 1.63 mol),4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)morpholine (592g, 1.95 mol), 1,4-dioxane (3.86 L), sodium carbonate (618 g, 5.83 mol),and water (771 ml) was degassed by bubbling nitrogen through the mixtureat 20° C. for 20 minutes and treated withtetrakis(triphenylphosphine)palladium(0) (14.11 g, 12.21 mmol). Theresulting mixture was degassed for an additional 20 minutes and thenheated to 87-89° C. for 17 h. After cooling to 20° C., the mixture wasdiluted with ethyl acetate (5.80 L) and a solution of(R)-2-Amino-3-mercaptopropionic acid (232 g) in water (2.320 L). Afterstirring for 1 h at 20° C., the organic layer was separated and washedagain with a solution of (R)-2-Amino-3-mercaptopropionic acid (232 g) inwater (2.320 L). The aqueous layers were combined and extracted withethyl acetate (5.80 L). The organic layers were combined, washed with asolution of sodium hydroxide (93 g) in water (2.32 L), and concentratedunder vacuum at 35° C. to give the title compound as an orange oil (1.21kg, 164% yield).

5-(Ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxylicacid (5): Methyl5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxylate(69.0 g, 152.5 mmol) (based on the theoretical yield from the previousstep) was suspended in ethanol (380 mL) and treated with a solution ofsodium hydroxide (24.84 g, 621.0 mmol) in water (207 mL). The mixturewas stirred at 40° C. for 18 h. After cooling to 0-5° C., the mixturewas neutralized to pH 6.5 with 1 N hydrochloric acid (580 mL)maintaining the temperature below 25° C. Then, the mixture was extractedtwice with a mixture of dichloromethane (690 mL) and methanol (69.0 mL).The organic layers were combined and concentrated under vacuum to give acrude product as a yellow solid (127 g).

The crude product was dissolved in 2-methyltetrahydrofuran (656 mL) at70° C. and then treated with IPA (828 mL). The mixture was allowed tocool to rt over 3-4 h and then stirred overnight at rt. The precipitatewas filtered, washed twice with IPA (207 mL), and dried under vacuum togive the title compound as an off white solid (53.54 g, 80%). The XRPDpattern of this compound is shown in FIG. 9.

N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide(Compound I): A mixture of5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxylicacid (540 g, 1.23 mol) and3-(aminomethyl)-4,6-dimethyl-dihydro-pyridin-2(1H)-one hydrochloride(279 g, 1.48 mol) was suspended in DMSO (2.70 L) and treated withtriethylamine (223 ml, 1.60 mol). The mixture was stirred at 25° C. for30 min and treated with EDC-HCl (354 g, 1.85 mol) and HOBT hydrate (283g, 1.85 mol). The reaction mixture was stirred at rt for 16 h. Afteraddition of triethylamine (292 ml, 2.09 mol), the mixture was cooled to15° C., diluted with water (10.1 L) maintaining the temperature below30° C., and stirred at 19-25° C. for 4 h. The resulting precipitate wasfiltered, washed twice with water (2.70 L), and dried under vacuum togive a crude product (695 g, wt-wt analysis=78%).

For the further purification of the product, recrystallization wasconducted. A crude product (20.00 g, 34.92 mmol) was suspended in amixture of ethanol (190 ml) and water (10.00 ml) and heated to 75° C.until a clear solution was obtained. The solution was allowed to cool tort overnight. The precipitate was filtered, washed twice with a mixtureof ethanol (30.0 ml) and water (30.0 ml), and dried under vacuum at 35°C. to give the title compound as an off white solid (14.0 g, 70%recovery from the crude and 90% yield based on wt-wt assay).

4-((3′-(((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)carbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methyl-[1,1′-biphenyl]-4-yl)methyl)morpholin-4-iumbromide (Polymorph A): A crudeN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide(595 g, 464 g based on wt-wt assay, 810.3 mmol) was suspended in ethanol(3.33 L). After heating to 70° C., the mixture was treated with 48%aqueous HBr (97 ml, 850.8 mmol) and stirred at 70° C. for 30 min. Theresulting orange-red solution was treated with ethyl acetate (3.33 L)maintaining the temperature above 60° C. The mixture was slowly cooledto rt over 18 h. The mixture was cooled to 0° C. over 1 h and stirred atthat temperature for 5.5 h. The resulting precipitate was filtered,washed twice with ethyl acetate (1.39 L), and dried under vacuum to givethe title compound as an off white solid (515 g, 97% yield).

Recrystallization of Polymorph A:4-((3′-(((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)carbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methyl-[1,1′-biphenyl]-4-yl)methyl)morpholin-4-iumbromide (0.50 g, 0.77 mmol; 95.6% pure by HPLC) was suspended in ethanol(3.0 mL) and heated to 80° C. until a clear solution was obtained. Tothe solution was added MTBE (5.0 mL) slowly. The resulting solution wasallowed to cool to 18-22° C. over 3 h and stirred at 18-22° C. for 15 h.The precipitate was filtered, washed twice with MTBE (2 mL) and driedunder vacuum to give 0.45 g of the title compound (89% recovery, 96.6%pure by HPLC). The X-ray powder diffraction pattern of Polymorph A(monohydrobromide) is shown in FIG. 1. Table 1, below, lists the mostsignificant peaks.

TABLE 1 Peaks (Degrees 2-theta) 3.9 10.1 14.3 17.5 18.7 20.6 20.9 21.822.0 23.3 23.6

Assessment of Hydrobromide of Compound I and Polymorph A

A number of different salt forms of Compound I were prepared andscreened, including hydrochloride, hydrobromide, hemisulfate, sodium,phosphate, nitrate, maleate, malonate, and L-tartrate salts. Among them,the hydrobromide (HBr) salt had the most advantageous physicochemicalproperties in terms of ease of preparation and hygroscopicity.

Detailed studies of the free base of Compound I as well as the HCl saltof this compound were carried out. At least five different crystal formswere detected from the free form of Compound I during preliminarypolymorph screening using XRD and DSC. Due to the high degree ofvariability observed during crystallization of the free form, crystalforms of other salts were pursued. Of the screened salts, themonohydrochloride, monohydrobromide, hemisulfate, phosphate, maleate,L-tartarate and sodium salt forms were crystalline. The phosphate andmaleate salts were very hygroscopic and L-tartarate had poorcrystallinity.

It was difficult to obtain high degree of crystallinity from HCl salt ofCompound I. A mixture of crystalline and amorphous material was obtainedirrespective of crystallization conditions. As shown in FIG. 8, DSC dataof the monohydrochloride salt of Compound I indicates some degree ofnon-crystallinity with an endotherm at 190.5° C. Also, dynamic vaporsorption (DVS) data for the monohydrochloride salt of Compound I wasobtained and found to show some hygroscopicity: between 4-6% weight gainwas observed at 75% relative humidity (RH) at 25° C. (FIG. 18B). Thismay be attributed to a certain amount of non-crystalline nature of themonohydrochloride salt. See, e.g., FIG. 18A, which shows an amorphoustrihydrochloride Compound I. Because the level of crystallinity was notcontrollable, the HCl salt was not considered for further development.

As shown in FIG. 6, DVS analysis of the sodium salt of Compound I showedsignificant hygroscopicity: approximately 15% weight gain was observedat 75% relative humidity (RH) at 25° C. As shown in FIG. 7, thehemisulfate salt of Compound I showed moderately high hygroscopicity:between 9-11% weight gain was observed at 75% relative humidity (RH) at25° C. This may be attributed to the highly non-crystalline nature ofthe compound, as DSC data of the hemisulfate salt indicates very highdegree of non-crystallinity with no clean endotherm.

Of these crystalline compounds, the monohydrobromide was the mostcrystalline and least hydroscopic (see FIGS. 1, 3, and 4). Furthermore,the monohydrobromide is highly stable, and resists generation ofimpurities (FIG. 5 depicts HPLC analysis of Polymorph A over three daysat an elevated temperature. Polymorph A produced minimal impurities overthree days 100° C.). Interestingly, the di-HBr salt of Compound I wasfound to be primarily amorphous (FIG. 2).

Two different crystal forms of the monohydrobromide of Compound I(Polymorphs A and B) were obtained from different solvent systems andcharacterized using XRD, DSC and TGA-DSC analyses. XRD and DSC data forthese two different crystal forms from representative batches ofCompound I are shown in FIG. 1, FIG. 3, and FIG. 10. Polymorph B ischaracterized by a powder XRD pattern with peaks at 8.5, 10.9, 16.7,17.4, 20.9, 22.1 and 25.7±0.2 degrees 2 theta (see FIG. 10). Betweenthese two, Polymorph A was found to be more crystalline in nature.Dynamic vapor adsorption (DVS) studies showed that the polymorph A isnon-hygroscopic (FIG. 4). In the thermal analyses, a single endothermicpeak was observed with an onset temperature approximately at 251° C. Inaddition, it was evident from DSC analysis that the recrystallization ofpolymorph A significantly increases the crystallinity of the material(see FIG. 3).

In multiple laboratory scale runs, Polymorph A was obtainedreproducibly, and slight changes in crystallization conditions did notresult in different crystal forms.

Wild-Type and Mutant PRC2 Enzyme Assays

General Materials. S-adenosylmethionine (SAM), S-adenosylhomocyteine(SAH), bicine, KCl, Tween20, dimethylsulfoxide (DMSO) and bovine skingelatin (BSG) were purchased from Sigma-Aldrich at the highest level ofpurity possible. Dithiothreitol (DTT) was purchased from EMD. ³H-SAM waspurchased from American Radiolabeled Chemicals with a specific activityof 80 Ci/mmol. 384-well streptavidin Flashplates were purchased fromPerkinElmer.

Substrates. Peptides representative of human histone H3 residues 21-44containing either an unmodified lysine 27 (H3K27me0) or dimethylatedlysine 27 (H3K27me2) were synthesized with a C-terminal G(K-biotin)linker-affinity tag motif and a C-terminal amide cap by 21^(st) CenturyBiochemicals. The peptides were high-performance liquid chromatography(HPLC) purified to greater than 95% purity and confirmed by liquidchromatography mass spectrometry (LC-MS). The sequences are listedbelow.

H3K27me0: (SEQ ID NO: 1) ATKAARKSAPATGGVKKPHRYRPGGK(biotin)-amideH3K27me2: (SEQ ID NO: 2) ATKAARK(me2)SAPATGGVKKPHRYRPGGK(biotin)-amide

Chicken erythrocyte oligonucleosomes were purified from chicken bloodaccording to established procedures.

Recombinant PRC2 Complexes. Human PRC2 complexes were purified as4-component enzyme complexes co-expressed in Spodoptera frugiperda (sf9)cells using a baculovirus expression system. The subunits expressed werewild-type EZH2 (NM_004456) or EZH2 Y641F, N, H, S or C mutants generatedfrom the wild-type EZH2 construct, EED (NM_003797), Suz12 (NM_015355)and RbAp48 (NM_005610). The EED subunit contained an N-terminal FLAG tagthat was used to purify the entire 4-component complex from sf9 celllysates. The purity of the complexes met or exceeded 95% as determinedby SDS-PAGE and Agilent Bioanalyzer analysis. Concentrations of enzymestock concentrations (generally 0.3-1.0 mg/mL) was determined using aBradford assay against a bovine serum albumin (BSA) standard.

General Procedure for PRC2 Enzyme Assays on Peptide Substrates. Theassays were all performed in a buffer consisting of 20 mM bicine(pH=7.6), 0.5 mM DTT, 0.005% BSG and 0.002% Tween20, prepared on the dayof use. Compounds in 100% DMSO (1 μL) were spotted into polypropylene384-well V-bottom plates (Greiner) using a Platemate 2×3 outfitted witha 384-channel pipet head (Thermo). DMSO (1 μL) was added to columns 11,12, 23, 24, rows A-H for the maximum signal control, and SAH, a knownproduct and inhibitor of PRC2 (1 μL) was added to columns 11, 12, 23,24, rows I-P for the minimum signal control. A cocktail (40 μL)containing the wild-type PRC2 enzyme and H3K27me0 peptide or any of theY641 mutant enzymes and H3K27me2 peptide was added by Multidrop Combi(Thermo). The compounds were allowed to incubate with PRC2 for 30 min at25° C., then a cocktail (10 μL) containing a mixture of non-radioactiveand ³H-SAM was added to initiate the reaction (final volume=51 μL). Inall cases, the final concentrations were as follows: wild-type or mutantPRC2 enzyme was 4 nM, SAH in the minimum signal control wells was 1 mMand the DMSO concentration was 1%. The final concentrations of the restof the components are indicated in Table 2, below. The assays werestopped by the addition of non-radioactive SAM (10 μL) to a finalconcentration of 600 μM, which dilutes the ³H-SAM to a level where itsincorporation into the peptide substrate is no longer detectable. 50 μLof the reaction in the 384-well polypropylene plate was then transferredto a 384-well Flashplate and the biotinylated peptides were allowed tobind to the streptavidin surface for at least lh before being washedthree times with 0.1% Tween20 in a Biotek ELx405 plate washer. Theplates were then read in a PerkinElmer TopCount platereader to measurethe quantity of ³H-labeled peptide bound to the Flashplate surface,measured as disintegrations per minute (dpm) or alternatively, referredto as counts per minute (cpm).

TABLE 2 Final concentrations of components for each assay variationbased upon EZH2 identity (wild-type or Y641 mutant EZH2) PRC2 Enzyme(denoted by Peptide Non-radioactive SAM ³H-SAM EZH2 identity) (nM) (nM)(nM) Wild-type 185 1800 150 Y641F 200 850 150 Y641N 200 850 150 Y641H200 1750 250 Y641S 200 1300 200 Y641C 200 3750 250

General Procedure for Wild-Type PRC2 Enzyme Assay on OligonucleosomeSubstrate. The assays was performed in a buffer consisting of 20 mMbicine (pH=7.6), 0.5 mM DTT, 0.005% BSG, 100 mM KCl and 0.002% Tween20,prepared on the day of use. Compounds in 100% DMSO (1 μL) were spottedinto polypropylene 384-well V-bottom plates (Greiner) using a Platemate2×3 outfitted with a 384-channel pipet head (Thermo). DMSO (1 μL) wasadded to columns 11, 12, 23, 24, rows A-H for the maximum signalcontrol, and SAH, a known product and inhibitor of PRC2 (1 μL) was addedto columns 11, 12, 23, 24, rows I-P for the minimum signal control. Acocktail (40 μL) containing the wild-type PRC2 enzyme and chickenerythrocyte oligonucleosome was added by Multidrop Combi (Thermo). Thecompounds were allowed to incubate with PRC2 for 30 min at 25° C., thena cocktail (10 μL) containing a mixture of non-radioactive and ³H-SAMwas added to initiate the reaction (final volume=51 μL). The finalconcentrations were as follows: wild-type PRC2 enzyme was 4 nM,non-radioactive SAM was 430 nM, ³H-SAM was 120 nM, chicken erythrocyteolignonucleosome was 120 nM, SAH in the minimum signal control wells was1 mM and the DMSO concentration was 1%. The assay was stopped by theaddition of non-radioactive SAM (10 μL) to a final concentration of 600μM, which dilutes the ³H-SAM to a level where its incorporation into thechicken erythrocyte olignonucleosome substrate is no longer detectable.50 μL of the reaction in the 384-well polypropylene plate was thentransferred to a 384-well Flashplate and the chicken erythrocytenucleosomes were immobilized to the surface of the plate, which was thenwashed three times with 0.1% Tween20 in a Biotek ELx405 plate washer.The plates were then read in a PerkinElmer TopCount platereader tomeasure the quantity of ³H-labeled chicken erythrocyte oligonucleosomebound to the Flashplate surface, measured as disintegrations per minute(dpm) or alternatively, referred to as counts per minute (cpm).

% Inhibition Calculation

${\%\mspace{14mu}{inh}} = {100 - {\left( \frac{{dpm}_{cmpd} - {dpm}_{m\; i\; n}}{{dpm}_{{ma}\; x} - {dpm}_{m\; i\; n}} \right) \times 100}}$

Where dpm=disintegrations per minute, cmpd=signal in assay well, and minand max are the respective minimum and maximum signal controls.

Four-Parameter IC₅₀ Fit

$Y = {{Bottom} + \frac{\left( {{Top} - {Bottom}} \right)}{1 + \left( \frac{X}{{IC}_{50}} \right)^{{Hill}\mspace{14mu}{Coefficient}}}}$

Where top and bottom are the normally allowed to float, but may be fixedat 100 or 0 respectively in a 3-parameter fit. The Hill Coefficientnormally allowed to float but may also be fixed at 1 in a 3-parameterfit. Y is the % inhibition and X is the compound concentration.

IC₅₀ values for the PRC2 enzyme assays on peptide substrates (e.g., EZH2wild type and Y641F) are presented in Table 3 below.

WSU-DLCL2 Methylation Assay

WSU-DLCL2 suspension cells were purchased from DSMZ (German Collectionof Microorganisms and Cell Cultures, Braunschweig, Germany).RPMI/Glutamax Medium, Penicillin-Streptomycin, Heat Inactivated FetalBovine Serum, and D-PBS were purchased from Life Technologies, GrandIsland, N.Y., USA. Extraction Buffer and Neutralization Buffer (5×) werepurchased from Active Motif, Carlsbad, Calif., USA. Rabbit anti-HistoneH3 antibody was purchased from Abcam, Cambridge, Mass., USA. Rabbitanti-H3K27me3 and HRP-conjugated anti-rabbit-IgG were purchased fromCell Signaling Technology, Danvers, Mass., USA. TMB “Super Sensitive”substrate was sourced from BioFX Laboratories, Owings Mills, Md., USA.IgG-free Bovine Serum Albumin was purchased from Jackson ImmunoResearch,West Grove, Pa., USA. PBS with Tween (10× PBST) was purchased from KPL,Gaithersburg, Md., USA. Sulfuric Acid was purchased from Ricca Chemical,Arlington, Tex., USA. Immulon ELISA plates were purchased from Thermo,Rochester, N.Y., USA. V-bottom cell culture plates were purchased fromCorning Inc., Corning, N.Y., USA.V-bottom polypropylene plates werepurchased from Greiner Bio-One, Monroe, N.C., USA.

WSU-DLCL2 suspension cells were maintained in growth medium (RPMI 1640supplemented with 10% v/v heat inactivated fetal bovine serum and 100units/mL penicillin-streptomycin) and cultured at 37° C. under 5% CO₂.Under assay conditions, cells were incubated in Assay Medium (RPMI 1640supplemented with 20% v/v heat inactivated fetal bovine serum and 100units/mL penicillin-streptomycin) at 37° C. under 5% CO₂ on a plateshaker.

WSU-DLCL2 cells were seeded in assay medium at a concentration of 50,000cells per mL to a 96-well V-bottom cell culture plate with 200 μL perwell. Compound (1 μL) from 96 well source plates was added directly toV-bottom cell plate. Plates were incubated on a titer-plate shaker at37° C., 5% CO2 for 96 hours. After four days of incubation, plates werespun at 241×g for five minutes and medium was aspirated gently from eachwell of cell plate without disturbing cell pellet. Pellet wasresuspended in 200 μL DPBS and plates were spun again at 241×g for fiveminutes. The supernatant was aspirated and cold (4° C.) Extractionbuffer (100 μL) was added per well. Plates were incubated at 4° C. onorbital shaker for two hours. Plates were spun at 3427×g×10 minutes.Supernatant (80 μL per well) was transferred to its respective well in96 well V-bottom polypropylene plate. Neutralization Buffer 5× (20 μLper well) was added to V-bottom polypropylene plate containingsupernatant. V-bottom polypropylene plates containing crude histonepreparation (CHP) were incubated on orbital shaker x five minutes. CrudeHistone Preparations were added (2 μL per well) to each respective wellinto duplicate 96 well ELISA plates containing 100 μL Coating Buffer (1×PBS+BSA 0.05% w/v). Plates were sealed and incubated overnight at 4° C.The following day, plates were washed three times with 300 μL per well1× PBST. Wells were blocked for two hours with 300 μL per well ELISADiluent ((PBS (1×) BSA (2% w/v) and Tween20 (0.05% v/v)). Plates werewashed three times with 1× PBST. For the Histone H3 detection plate, 100μL per well were added of anti-Histone-H3 antibody (Abcam, ab1791)diluted 1:10,000 in ELISA Diluent. For H3K27 trimethylation detectionplate, 100 μL per well were added of anti-H3K27me3 diluted 1:2000 inELISA diluent. Plates were incubated for 90 minutes at room temperature.Plates were washed three times with 300 μL 1× PBST per well. For HistoneH3 detection, 100 μL of HRP-conjugated anti-rabbit IgG antibody dilutedto 1:6000 in ELISA diluent was added per well. For H3K27me3 detection,100 μL of HRP conjugated anti-rabbit IgG antibody diluted to 1:4000 inELISA diluent was added per well. Plates were incubated at roomtemperature for 90 minutes. Plates were washed four times with 1× PBST300 μL per well. TMB substrate100 μL was added per well. Histone H3plates were incubated for five minutes at room temperature. H3K27me3plates were incubated for 10 minutes at room temperature. The reactionwas stopped with sulfuric acid 1N (100 μL per well). Absorbance for eachplate was read at 450 nm.

First, the ratio for each well was determined by:

$\left( \frac{H\; 3K\; 27{me}\; 3\mspace{14mu}{OD}\; 450}{{Histone}\mspace{14mu} H\; 3\mspace{14mu}{OD}\; 450} \right).$

Each plate included eight control wells of DMSO only treatment (MinimumInhibition) as well as eight control wells for maximum inhibition(Background wells).

The average of the ratio values for each control type was calculated andused to determine the percent inhibition for each test well in theplate. Test compound was serially diluted three-fold in DMSO for a totalof ten test concentrations, beginning at 25 μM. Percent inhibition wasdetermined and IC₅₀ curves were generated using duplicate wells perconcentration of compound. IC₅₀ values for this assay are presented inTable 3 below.

${{Percent}\mspace{14mu}{Inhibition}} = {100 - \left( {\left( \frac{\left( {{Individual}\mspace{14mu}{Test}\mspace{14mu}{Sample}\mspace{14mu}{Ratio}} \right) - \left( {{Background}\mspace{14mu}{Avg}\mspace{14mu}{Ratio}} \right)}{\left( {{Minimum}\mspace{14mu}{Inhibition}\mspace{14mu}{Ratio}} \right) - \left( {{Background}\mspace{14mu}{Average}\mspace{14mu}{Ratio}} \right)} \right)*100} \right)}$

Cell Proliferation Analysis

WSU-DLCL2 suspension cells were purchased from DSMZ (German Collectionof Microorganisms and Cell Cultures, Braunschweig, Germany).RPMI/Glutamax Medium, Penicillin-Streptomycin, Heat Inactivated FetalBovine Serum were purchased from Life Technologies, Grand Island, N.Y.,USA. V-bottom polypropylene 384-well plates were purchased from GreinerBio-One, Monroe, N.C., USA. Cell culture 384-well white opaque plateswere purchased from Perkin Elmer, Waltham, Mass., USA. Cell-Titer Glogwas purchased from Promega Corporation, Madison, Wis., USA. SpectraMaxM5 plate reader was purchased from Molecular Devices LLC, Sunnyvale,Calif., USA.

WSU-DLCL2 suspension cells were maintained in growth medium (RPMI 1640supplemented with 10% v/v heat inactivated fetal bovine serum andcultured at 37° C. under 5% CO2. Under assay conditions, cells wereincubated in Assay Medium (RPMI 1640 supplemented with 20% v/v heatinactivated fetal bovine serum and 100 units/mL penicillin-streptomycin)at 37° C. under 5% CO2.

For the assessment of the effect of compounds on the proliferation ofthe WSU-DLCL2 cell line, exponentially growing cells were plated in384-well white opaque plates at a density of 1250 cell/ml in a finalvolume of 50 μl of assay medium. A compound source plate was prepared byperforming triplicate nine-point 3-fold serial dilutions in DMSO,beginning at 10 mM (final top concentration of compound in the assay was20 μM and the DMSO was 0.2%). A 100 nL aliquot from the compound stockplate was added to its respective well in the cell plate. The 100%inhibition control consisted of cells treated with 200 nM finalconcentration of staurosporine and the 0% inhibition control consistedof DMSO treated cells. After addition of compounds, assay plates wereincubated for 6 days at 37° C., 5% CO₂, relative humidity>90% for 6days. Cell viability was measured by quantization of ATP present in thecell cultures, adding 35 μl of Cell Titer Glog reagent to the cellplates. Luminescence was read in the SpectraMax M5. The concentrationinhibiting cell viability by 50% was determined using a 4-parametric fitof the normalized dose response curves. IC₅₀ values for this assay arepresented in Table 3 below.

TABLE 3 WSU WSU EZH2 IC50 Y641F prolif ELISA peptide v2 IC50 IC50 IC50Compound I 0.01299 0.01107 0.369 0.29 (free base)

In Vivo Study—SUDHL10 Human Lymphoma Cell Line

Mice

Female Fox Chase SCID® Mice (CB17/Icr-Prkdcscid/IcrIcoCrl, BeijingVitalriver Laboratory Animal Co., LTD) were 6-8 weeks old and had abody-weight (BW) range of 16.0-21.1 g on D1 of the study. The animalswere fed ad libitum with water (sterile) and irradiation sterilized drygranule food. The mice were housed on corn cob bedding in staticmicroisolators on a 12-hour light cycle at 20-22° C. (68-72° F.) and40-60% humidity. All procedures comply with the recommendations of theGuide for Care and Use of Laboratory Animals with respect to restraint,husbandry, surgical procedures, feed and fluid regulation, andveterinary care.

Tumor Cell Culture

Human lymphoma cell line SUDHL10 was obtained from DSMZ and maintainedat the CRO as suspension cultures in RPMI-1640 medium containing 100units/mL penicillin G sodium salt, 100 g/mL streptomycin and 10% fetalbovine serum. The cells were cultured in tissue culture flasks in ahumidified incubator at 37° C., in an atmosphere of 5% CO2 and 95% air.Only cultures below passage 12 were used for implantation

In Vivo Tumor Implantation

SUDHL10 human lymphoma cell line was harvested during mid-log phasegrowth, and re-suspended in PBS with 50% Matrigel™ (BD Biosciences).Each mouse received 1×10⁷ cells (0.2 mL cell suspension) subcutaneouslyin the right flank. Tumors were calipered in two dimensions to monitorgrowth as the mean volume approached the desired 80-120 mm³ range. Tumorsize, in mm³, was calculated from:

${{Tumor}\mspace{14mu}{Volume}} = \frac{w^{2} \times l}{2}$where w=width and l=length, in mm, of the tumor. Tumor weight can beestimated with the assumption that 1 mg is equivalent to 1 mm³ of tumorvolume. After 10 days mice with 72-256 mm³ tumors were sorted into fourgroups (n=16 per group) with mean tumor volumes of 173-179 mm³.Test Articles

The hydrobromide of Compound I was stored at room temperature andprotected from light. On each treatment day, a fresh compoundformulations were prepared by suspending the powder in 0.5% sodiumcarboxymethylcellulose (NaCMC) and 0.1% Tween® 80 in deionized water.The vehicle, 0.5% NaCMC and 0.1% Tween® 80 in deionized water, was usedto treat the control group at the same schedule. Formulations werestored away from light at 4° C. prior to administration.

Treatment Plan

Mice were treated with doses of the hydrobromide of Compound I rangingfrom 125-500 mg/kg and at a BID (twice a day every 12 h) schedules for28 days by oral gavage. Each dose was delivered in a volume of 0.2 mL/20g mouse (10 mL/kg), and adjusted for the last recorded weight ofindividual animals. On day 25 the 8 mice with the smallest tumors pergroup were chosen for a tumor growth delay endpoint (observation up to60 days). The remaining animals were euthanized on day 28 3 h after thelast dose for tumor collection.

Median Tumor Volume (MTV) and Tumor Growth Inhibition (TGI) Analysis

Treatment efficacy was determined on the last treatment day. MTV(n), themedian tumor volume for the number of animals, n, evaluable on the lastday, was determined for each group. Percent tumor growth inhibition (%TGI) can be defined several ways. First, the difference between theMTV(n) of the designated control group and the MTV(n) of thedrug-treated group is expressed as a percentage of the MTV(n) of thecontrol group:

${\%\mspace{14mu}{TGI}} = {\left( \frac{{{MTV}(n)}_{control} - {{MTV}(n)}_{treated}}{{{MTV}(n)}_{control}} \right) \times 100}$Another way of calculating % TGI is taking the change of the tumor sizefrom day 1 to day n into account with n being the last treatment day.

${\%\mspace{14mu}{TGI}} = {\left( \frac{{\Delta\;{{MTV}(n)}_{control}} - {\Delta\;{{MTV}(n)}_{treated}}}{\Delta\;{MTV}_{control}} \right) \times 100}$Δ MTV_(control) = MTV(n)_(control) − MTV(1)_(control)Δ MTV_(treated) = MTV(n)_(treated) − MTV(1)_(treated)Tumor Growth Delay Analysis

Eight mice per group were kept alive after the last treatment day fortumor growth delay analysis. Tumors were callipered twice-weekly andeach test animal was euthanized when its neoplasm reached the endpointvolume of 2000 mm³ or on the pre-specified last day of the study,whichever came first. Kaplan Meier survival analysis was performed.

Toxicity

Animals were weighed daily on Days 1-5, and then twice weekly until thecompletion of the study. The mice were examined frequently for overtsigns of any adverse, treatment related side effects, which weredocumented. Acceptable toxicity for the maximum tolerated dose (MTD) wasdefined as a group mean BW loss of less than 20% during the test, andnot more than 10% mortality due to TR deaths. A death was to beclassified as TR if it was attributable to treatment side effects asevidenced by clinical signs and/or necropsy, or due to unknown causesduring the dosing period. A death was to be classified as NTR if therewas evidence that the death was unrelated to treatment side effects. NTRdeaths during the dosing interval would typically be categorized as NTRa(due to an accident or human error) or NTRm (due to necropsy-confirmedtumor dissemination by invasion and/or metastasis). Orally treatedanimals that die from unknown causes during the dosing period may beclassified as NTRu when group performance does not support a TRclassification and necropsy, to rule out a dosing error, is notfeasible.

Sampling

On day 28 eight mice with the largest tumors were sampled in apre-specified fashion to assess target inhibition in tumors. Tumors wereharvested from specified mice under RNAse free conditions and bisected.Total tumor weight was measured. Frozen tumor tissue from each animalwas snap frozen in liquid N₂ and pulverized with a mortar and pestle.

Statistical and Graphical Analyses

All statistical and graphical analyses were performed with Prism 3.03(GraphPad) for Windows. To test statistical significance between thecontrol and treated groups over the whole treatment time course arepeated measures ANOVA test followed by Dunnets multiple comparisonpost test was employed. Prism reports results as non-significant (ns) atP>0.05, significant (symbolized by “*”) at 0.01<P<0.05, very significant(“**”) at 0.001<P<0.01 and extremely significant (“***”) at P<0.001. Forthe tumor growth delay arm of the study the percentage of animals ineach group remaining in the study versus time was presented in aKaplan-Meier survival plot.

Histone Extraction

For isolation of histones, 60-90 mg tumor tissue was homogenized in 1.5ml nuclear extraction buffer (10 mM Tris-HCl, 10 mM MgCl2, 25 mM KCl, 1%Triton X-100, 8.6% Sucrose, plus a Roche protease inhibitor tablet1836145) and incubated on ice for 5 minutes. Nuclei were collected bycentrifugation at 600 g for 5 minutes at 4° C. and washed once in PBS.Supernatant was removed and histones extracted for one hour, withvortexing every 15 minutes, with 0.4 N cold sulfuric acid. Extracts wereclarified by centrifugation at 10000 g for 10 minutes at 4° C. andtransferred to a fresh microcentrifuge tube containing 10× volume of icecold acetone. Histones were precipitated at −20° C. for 2hours-overnight, pelleted by centrifugation at 10000 g for 10 minutesand resuspended in water.

ELISA

Histones were prepared in equivalent concentrations in coating buffer(PBS+0.05% BSA) yielding 0.5 ng/ul of sample, and 100 ul of sample orstandard was added in duplicate to 2 96-well ELISA plates (ThermoLabsystems, Immulon 4HBX #3885). The plates were sealed and incubatedovernight at 4° C. The following day, plates were washed 3× with 300ul/well PBST (PBS+0.05% Tween 20; 10× PBST, KPL #51-14-02) on a Bio Tekplate washer. Plates were blocked with 300 ul/well of diluent (PBS+2%BSA+0.05% Tween 20), incubated at RT for 2 hours, and washed 3× withPBST. All antibodies were diluted in diluent. 100 ul/well ofanti-H3K27me3 (CST #9733, 50% glycerol stock 1:1,000) or anti-total H3(Abcam ab1791, 50% glycerol 1:10,000) was added to each plate. Plateswere incubated for 90 min at RT and washed 3× with PBST. 100 ul/well ofanti-Rb-IgG-HRP (Cell Signaling Technology, 7074) was added 1:2,000 tothe H3K27Me3 plate and 1:6,000 to the H3 plate and incubated for 90 minat RT. Plates were washed 4× with PBST. For detection, 100 ul/well ofTMB substrate (BioFx Laboratories, # TMBS) was added and platesincubated in the dark at RT for 5 min. Reaction was stopped with 100ul/well 1N H₂SO₄. Absorbance at 450 nm was read on SpectaMax M5Microplate reader.

Results:

Mice bearing SUDHL10 tumor xenografts were treated with the hydrobromideof Compound I at the maximal tolerated dose of 500 mg/kg BID andfractions of the MTD (½ and ¼ MTD). All doses were well tolerated for 28days without any significant body weight loss. There was onenon-treatment related death in the 500 mg/kg group on day 15 due to adosing error. All doses resulted in tumor growth inhibition whencompared to vehicle on day 28 (Table 4), and the 250 mg/kg and 500 mg/kgBID groups induced regressions (TGI>100%).

TABLE 4 Summary of tumor growth inhibition values induced byhydrobromide of Compound I in SUDHL10 xenografts Group % TGI from day 1% TGI from day 8 125 mg/kg BID 54 57 250 mg/kg BID 101 113 500 mg/kg BID104 115

FIG. 12A shows the growth of the SUDHL10 xenograft tumors over time forthe different treatment groups. The 125 mg/kg BID group was notsignificantly different from the vehicle group by repeated measuresANOVA and Dunnett's post test, but the mean terminal tumor size on day28 was significantly smaller than the one in the vehicle group (2 wayANOVA with Bonferroni post test, p<0.0001). Dosing of 250 mg/kg BID and500 mg/kg BID of the hydrobromide of Compound I for 28 days inducedcomparable regression responses as the terminal tumor weights on day 28were similar for those 2 groups (FIG. 12B).

Histones isolated from tumors harvested on day 28 (3 h after the lastdose) were subjected to ELISA analysis for global H3K27me3 levels. FIG.13 shows a clear dose dependent down-regulation of the H3K27me3 methylmark with treatment by the hydrobromide of Compound I. This figure showsglobal H3K27me3 methylation in SUDHL10 tumors from mice treated with thehydrobromide of Compound I for 28 days.

On day 25 eight mice per group with smallest tumors were chosen for atumor growth delay study to assess the re-growth of tumors after dosingstop on day 28. Mice were euthanized when their tumors reached a size of2000 mm³ or on day 60 (whichever comes first). These data were used toperform a Kaplan Meier survival analysis. FIG. 14A shows that tumorre-growth was clearly dose-dependent, and all mice treated with thehighest dose of 500 mg/kg BID for 28 days survived until day 60. Only 2mice had to be euthanized in the 250 mg/kg group before day 60. Mice inthe 125 mg/kg group had a clear survival benefit over vehicle treatedmice with an increase in median survival of 15.5 days (FIG. 14B).

Anti-Cancer Effect of the Hydrobromide of Compound I on the PfeifferHuman Diffused Large B-Cell Lymphoma Mouse Xenograft Model

The monohydrobromide of Compound I was tested for its anti-canceractivity in Pfeiffer mouse xenograft model, which is a human diffusedlarge B-Cell lymphoma xenograft model. Female of 5-week old NSG mice(Jackson Labs, Bar Harbor, Me.) were implanted subcutaneously with 20 to25 mg tumor fragments. Treatment was started approximately 31 days afterthe tumor implantation, when the average tumors reached approximately365 mm³. The treatment scheme is described in Table 5.

TABLE 5 Dosing Scheme No. of Route and Group Animals Treatment ScheduleA 9 Vehicle (0.5% Methyl Cellulose, PO; qdx28 0.1% Tween-80) B 9 34.2mg/kg Compound I (HBr salt) PO; qdx28 C 9 114 mg/kg Compound I (HBrsalt) PO; qdx28 D 9 342 mg/kg Compound I (HBr salt) PO; qdx28 E 9 1140mg/kg Compound I (HBr salt)  PO; qdx12^(§) ^(§)Due to compoundtolerability issue, only 12 daily doses were given to this group.

Tumor volume was followed throughout the experiment. Tumor volume wasmeasured two times weekly after the start of treatment. Tumor burden(mg=mm³) was calculated from caliper measurements by the formula for thevolume of a prolate ellipsoid (L×W²)/2 where L and W are the respectiveorthogonal length and width measurements (mm).

Day 1 was the day of the first treatment, and Day 28 was the day of thelast treatment. This study was terminated 36 days after the last dose,so Day 64 was the day of study termination. The primary endpoints usedto evaluate efficacy in this study were complete tumor regressions (CR),tumor sizes among groups, and percentage of tumor inhibition at the endof the study. A complete response was defined as a decrease in tumorsize to an undetectable size (<20 mm³) at the end of the study. Valuesfor percentage of tumor inhibition were calculated from the formula[1−(ΔT/ΔC)]×100, where ΔT and ΔC are changes in mean tumor volume (Agrowth) for each treated (T) and vehicle control group (C). T₀ and C₀(one day before the first dose) were used for the starting tumor volume.Additionally, tumor volumes which were taken one day after the last dose(T₂₉ and C₂₉) were used for the calculation of ΔT and ΔC. When the valuewas more than 100%, it was concluded as 100%. The formula used for thecalculation of percentage of tumor inhibition is shown below.

${{Percentage}\mspace{14mu}{of}\mspace{14mu}{tumor}\mspace{14mu}{inhibition}} = {\left\{ {1 - \frac{T_{29} - T_{0}}{C_{29} - C_{0}}} \right\} \times 100\%}$

During the treatment period, it was found that animals cannot toleratethe daily treatment of 1142 mg/kg the hydrobromide of Compound I andthree animals in this group (group E) required euthanasia after firstweek of treatment due to loss of more than 20% baseline bodyweight.Hence, drug administration for this group was stopped after 12 doses.Animals in other three dosing groups, except one animal in group D (342mg/kg hydrobromide of Compound I), all tolerated the 28-day treatmentwell with minimal bodyweight loss. Relative mouse body weight wasgraphed in FIG. 15. Animal bodyweight obtained on Day 0 was used as thebaseline bodyweight in the graph.

The hydrobromide of Compound I showed potent and long-lastinganti-cancer activity in Pfeiffer model with 100% of CR rate in three outof four dosing group (Table 6). Moreover, tumor re-growth was notobserved even 36 days after cessation of the treatment. This suggeststhat all the tumor cells were killed during the treatment. Althoughtumor re-growth was observed in the group with the lowest dose (group B,34.2 mg/kg), clear tumor stasis activity was observed during thetreatment period (FIG. 16). Tumor only started to grow upon cessation ofthe treatment (FIG. 16). This result also suggests that the tumor stasisactivity observed in group B is indeed test article-induced activity.

TABLE 6 Results summary table Percentage TV of tumor Group Treatment CR(Mean ± StDev) inhibition P value^(€) A Vehicle 0 2882 ± 2190 n/a n/a B34.2 mg/kg 0 497 ± 287  93% P < 0.05 Cmp I (HBr) C 114 mg/kg 9 0 100% P< 0.05 Cmp I (HBr) D 342 mg/kg  8^(£) 0 100% P < 0.05 Cmp I (HBr) E 1140mg/kg  6^(¥) 0 100% P < 0.05 Cmp I (HBr) ^(€)One way analysis ofvariance (ANOVA) followed by Dunnett's multiple comparison test (Prismsoftware version 5.02, Lake Forest, CA). ^(£)1 animal was euthanized onday 36 for low bodyweight. ^(¥)3 animals were euthanized on day 7, 9,and 11, individually for low bodyweight.

The invention claimed is:
 1. A polymorph ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidehydrobromide, wherein the polymorph exhibits an X-ray powder diffractionpattern having one or more characteristic peaks expressed in degrees2-theta at about 10.1+/−0.3 degrees, about 17.5+/−0.3 degrees, and about22.0+/−0.3 degrees 2-theta.
 2. The polymorph of to claim 1, wherein thepolymorph exhibits an X-ray powder diffraction pattern having one ormore characteristic peaks expressed in degrees 2-theta at about14.3+/−0.3 degrees, about 18.7+/−0.3 degrees, about 23.3+/−0.3 degrees,and about 23.6+/−0.3 degrees 2-theta.
 3. The polymorph of claim 1,wherein the polymorph exhibits an X-ray powder diffraction patternhaving at least 5 characteristic peaks expressed in degrees 2-theta atabout 10.1+/−0.3 degrees, about 14.3+/−0.3 degrees, about 17.5+/−0.3degrees, about 18.7+/−0.3 degrees, about 20.6+/−0.3 degrees, about20.9+/−0.3 degrees, about 21.8+/−0.3 degrees, about 22.0+/−0.3 degrees,about 23.3+/−0.3 degrees and about 23.6+/−0.3 degrees 2-theta.
 4. Thepolymorph of claim 1, wherein the polymorph exhibits an X-ray powderdiffraction pattern having at least 6 characteristic peaks expressed indegrees 2-theta at about 10.1+/−0.3 degrees, about 14.3+/−0.3 degrees,about 17.5+/−0.3 degrees, about 18.7+/−0.3 degrees, about 20.6+/−0.3degrees, about 20.9+/−0.3 degrees, about 21.8+/−0.3 degrees, about22.0+/−0.3 degrees, about 23.3+/−0.3 degrees and about 23.6+/−0.3degrees 2-theta.
 5. The polymorph of claim 1, wherein the polymorphexhibits an X-ray powder diffraction pattern having at least 7characteristic peaks expressed in degrees 2-theta at about 10.1+/−0.3degrees, about 14.3+/−0.3 degrees, about 17.5+/−0.3 degrees, about18.7+/−0.3 degrees, about 20.6+/−0.3 degrees, about 20.9+/−0.3 degrees,about 21.8+/−0.3 degrees, about 22.0+/−0.3 degrees, about 23.3+/−0.3degrees and about 23.6+/−0.3 degrees 2-theta.
 6. The polymorph of claim1, wherein the polymorph exhibits an X-ray powder diffraction patternhaving characteristic peaks expressed in degrees 2-theta at about10.1+/−0.3 degrees, about 14.3+/−0.3 degrees, about 17.5+/−0.3 degrees,about 18.7+/−0.3 degrees, about 20.6+/−0.3 degrees, about 20.9+/−0.3degrees, about 21.8+/−0.3 degrees, about 22.0+/−0.3 degrees, about23.3+/−0.3 degrees and about 23.6+/−0.3 degrees 2-theta.
 7. Thepolymorph of claim 1, wherein the polymorph exhibits a differentialscanning calorimetry thermogram having a characteristic peak expressedin units of ° C. at a temperature of 255+/−5° C.
 8. A pharmaceuticalcomposition comprising the polymorph of claim 1, and a pharmaceuticallyacceptable carrier or diluent.
 9. The pharmaceutical composition ofclaim 8, wherein the polymorph exhibits an X-ray powder diffractionpattern having one or more characteristic peaks expressed in degrees2-theta at about 14.3+/−0.3 degrees, about 18.7+/−0.3 degrees, about23.3+/−0.3 degrees, and about 23.6+/−0.3 degrees 2-theta.
 10. Thepharmaceutical composition of claim 8, wherein the polymorph exhibits anX-ray powder diffraction pattern having at least 5 characteristic peaksexpressed in degrees 2-theta at about 10.1+/−0.3 degrees, about14.3+/−0.3 degrees, about 17.5+/−0.3 degrees, about 18.7+/−0.3 degrees,about 20.6+/−0.3 degrees, about 20.9+/−0.3 degrees, about 21.8+/−0.3degrees, about 22.0+/−0.3 degrees, about 23.3+/−0.3 degrees and about23.6+/−0.3 degrees 2-theta.
 11. The pharmaceutical composition of claim8, wherein the polymorph exhibits an X-ray powder diffraction patternhaving at least 6 characteristic peaks expressed in degrees 2-theta atabout 10.1+/−0.3 degrees, about 14.3+/−0.3 degrees, about 17.5+/−0.3degrees, about 18.7+/−0.3 degrees, about 20.6+/−0.3 degrees, about20.9+/−0.3 degrees, about 21.8+/−0.3 degrees, about 22.0+/−0.3 degrees,about 23.3+/−0.3 degrees and about 23.6+/−0.3 degrees 2-theta.
 12. Thepharmaceutical composition of claim 8, wherein the polymorph exhibits anX-ray powder diffraction pattern having at least 7 characteristic peaksexpressed in degrees 2-theta at about 10.1+/−0.3 degrees, about14.3+/−0.3 degrees, about 17.5+/−0.3 degrees, about 18.7+/−0.3 degrees,about 20.6+/−0.3 degrees, about 20.9+/−0.3 degrees, about 21.8+/−0.3degrees, about 22.0+/−0.3 degrees, about 23.3+/−0.3 degrees and about23.6+/−0.3 degrees 2-theta.
 13. The pharmaceutical composition of claim8, wherein the polymorph exhibits an X-ray powder diffraction patternhaving characteristic peaks expressed in degrees 2-theta at about10.1+/−0.3 degrees, about 14.3+/−0.3 degrees, about 17.5+/−0.3 degrees,about 18.7+/−0.3 degrees, about 20.6+/−0.3 degrees, about 20.9+/−0.3degrees, about 21.8+/−0.3 degrees, about 22.0+/−0.3 degrees, about23.3+/−0.3 degrees and about 23.6+/−0.3 degrees 2-theta.
 14. Thepharmaceutical composition of claim 8, wherein the polymorph exhibits adifferential scanning calorimetry thermogram having a characteristicpeak expressed in units of ° C. at a temperature of 255+/−5° C.
 15. Thepolymorph of claim 1, wherein the polymorph is substantially free ofimpurities.
 16. The polymorph of claim 1, wherein the polymorph containsless than 5% by weight total impurities.
 17. The polymorph of claim 1,wherein the polymorph is a crystalline solid substantially free ofamorphousN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidehydrobromide.
 18. The pharmaceutical composition of claim 8, wherein thepolymorph is substantially free of impurities.
 19. The pharmaceuticalcomposition of claim 8, wherein the polymorph contains less than 5% byweight total impurities.
 20. The pharmaceutical composition of claim 8,wherein the polymorph is a crystalline solid substantially free ofamorphousN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidehydrobromide.